Mitochondrial transplantation improves bioenergetics and neurite outgrowth in disease-associated P301Ltau-expressing neuronal cells

Mitochondrial transplantation improves bioenergetics and neurite outgrowth in disease-associated P301Ltau-expressing neuronal cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mitochondrial transplantation improves bioenergetics and neurite outgrowth in disease-associated P301Ltau-expressing neuronal cells Aline Broeglin, Aurélien Riou, Andreas Papassotiropoulos, Anne Eckert, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6502413/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Dec, 2025 Read the published version in Molecular Neurobiology → Version 1 posted 10 You are reading this latest preprint version Abstract Tauopathies are neurodegenerative diseases characterized by the abnormal accumulation of tau protein in neurons, leading to cognitive impairment. A common feature of these disorders is mitochondrial dysfunction, which results in bioenergetic deficits and contributes to neuronal death. As neurons have high energy demands, impaired mitochondrial function directly affects their viability and function. Thus, mitochondria represent an attractive target for neuroprotective strategies in tauopathies. Mitochondrial transplantation (MT) is an emerging therapeutic approach to restoring cellular bioenergetics. Although MT has shown promise in various models of brain diseases, its efficacy has not been evaluated in the context of tau-induced mitochondrial dysfunction. This study investigates the therapeutic potential of MT in a cellular model of tauopathy. Mitochondria were freshly isolated from astrocytic cells and transplanted into healthy SH-SY5Y neuroblastoma cells and SH-SY5Y cells overexpressing the P301L tau mutation. Bioenergetic and neuroplastic parameters were assessed 24 and 48h post-transplantation. Our results demonstrate that MT enhances cell viability, ATP production, mitochondrial membrane potential, and respiration in both healthy and tau-mutant neuronal cells. In addition, MT reduced mitochondrial superoxide anion levels and promoted neurite outgrowth in both cell lines. These findings suggest that MT is a promising therapeutic strategy for tauopathies. Importantly, this approach positions mitochondria not as a target but as the therapeutic agent itself. Further studies are warranted to advance mitochondrial transplantation toward clinical applications in tau-related neurodegenerative disorders. Tauopathies P301Ltau mutation Mitochondria Transplantation Bioenergetic Neurites Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Neurodegenerative diseases are predominantly age-related and constitute a significant public health concern. These conditions are characterized by progressive degeneration and loss of neuronal cells in the central and peripheral nervous systems [ 1 , 25 ]. Tauopathies belong to this group of neurodegenerative diseases. Notable examples include Alzheimer's disease (AD), fronto-temporal dementia (FTD), progressive supranuclear palsy, and amyotrophic lateral sclerosis. The tau protein, which is encoded by the MAPT gene (Microtubule Associated Protein Tau) on chromosome 17, plays a crucial role in the stabilization, assembly, and function of microtubules [ 5 ]. Moreover, this protein facilitates several essential functions in neuronal cells, including axonal transport, neurotransmission, and cell polarity [ 32 ]. However, in a pathological context, tau protein undergoes an abnormal hyperphosphorylation, resulting in intracellular tau accumulation and neurofibrillary tangle formation in the brain. The mechanisms underlying tau-induced neuronal dysfunction are not fully understood. Studies have demonstrated that tau protein exhibits abnormal interactions with mitochondria, disrupting their cell functions and contributing to neuronal death and loss [ 14 , 32 ]. Mitochondria, also known as the "powerhouses of the cell," are essential organelles contributing to vital cellular functions. They are not only required to ensure the production of the primary cellular energy through the generation of adenosine triphosphate (ATP) but they are also involved in a wide range of functions, such as calcium buffering and metabolite synthesis [ 32 ]. Mitochondria are essential in neurons that require approximately 15% of the total body's energy to ensure processes such as neurotransmitter release, action potential, and synaptic plasticity [ 26 ]. Given the pivotal role of mitochondria in these post-mitotic and highly differentiated cells, the impact of mitochondrial defects on brain functions is a salient consideration. Indeed, in tauopathies, the abnormal interaction of mitochondria with tau protein leads to impairment in mitochondrial morphology, like mitochondria swelling, and a decrease in their bioenergetic functions, such as impaired ATP production and an increase in reactive oxygen species (ROS) generation [ 7 ]. Furthermore, mitochondrial dynamics, particularly the balance between fusion and fission processes, are altered, resulting in an impairment of the mitochondrial recycling system, also known as "mitophagy, which in turn leads to increased cellular stress [ 6 , 18 ]. These mitochondrial impairments impact several cellular functions, including neuroplasticity with a deficit observed in the neurite outgrowth process. Indeed, the hyperphosphorylated tau protein cannot stabilize the microtubules correctly, which is necessary for the neurite’s elongation. Besides, it has been shown that the physiological tau protein can potentiate some pathways involved in neurite elongation initiation [ 17 ]. It is widely recognized that these mitochondrial dysfunctions often precede the cognitive deficits observed in neurodegenerative diseases [ 28 ]. Neurons, as post-mitotic cells, cannot divide and replenish their supply in mitochondria [ 26 ]. Therefore, neuronal mitochondria represent an interesting target for therapeutic intervention. Conventional therapeutic approaches usually consist in improving mitochondrial bioenergetic functions and decreasing mitochondrial oxidative damage. A new approach consists of using exogenous healthy mitochondria as a treatment itself. This approach is called here mitochondrial transplantation. This innovative technique involves the isolation of mitochondria from healthy donor cells and their subsequent transfer into recipient cells that exhibit mitochondrial dysfunctions. This approach has been extensively documented in the context of cardiac diseases, where mitochondria also play a crucial role, as evidenced by the research conducted by the group of James McCully (Harvard Medical School, Boston, USA). They have demonstrated the efficacy of mitochondrial transplantation in enhancing cellular viability following ischemia-reperfusion injury in the heart [ 9 ]. An increasing number of research groups are exploring the potential of mitochondrial transplantation to treat disorders in various organs, including the kidneys, liver, and brain [ 19 ]. Mitochondrial transplantation has been tested in both in vitro and in vivo settings for several brain disorders, including traumatic brain injury, cognitive deficit, neurodegenerative diseases, and brain cancer (reviewed in [ 26 ]). Overall, encouraging results are observed after mitochondrial transplantation, with notably an increase in mitochondrial functions, cellular viability, and an improvement of cognitive performance [ 22 , 30 ]. However, no research has been conducted on the impact of mitochondrial transplantation in the context of tauopathies until today. To investigate this novel aspect, a cellular model (SH-SY5Y cells) overexpressing the P301L-tau mutation was used in the present study. This model has been previously characterized in our laboratory and has been shown to exhibit abnormal tau hyperphosphorylation, accompanied by bioenergetic deficits and mitochondrial impairments [ 15 , 31 ]. It has been demonstrated that physiological transfers between glial cells and neurons occur in physiological conditions, leading to neuroprotective effects and improvement of cellular functions. Therefore, an astrocytic cell line named “A172” was used as a donor of healthy mitochondria for the transplantation [ 11 ]. We hypothesized that mitochondrial transplantation could enhance cellular viability, neurite outgrowth, and bioenergetic functions in cells overexpressing disease-associated tau protein. In the first step, we investigated the effect of mitochondrial transplantation on a healthy cellular model to ascertain the optimal parameters. Namely, we assessed the impact of mitochondrial transplantation on cell viability, ATP levels, mitochondrial membrane potential, cellular oxygen consumption rate, and reactive oxygen species (ROS) levels. In the next step, we used the same approach and bioenergetic readouts with the "P301L-tau" cellular model of tauopathies. Finally, the study was completed with a neurite outgrowth investigation on the healthy and the P301L cells to assess the impact of mitochondria transplantation on neuronal morphology and differentiation. Materials and methods Reagents/Chemicals All the materials used for the study are listed in Table 1 . Table 1 List of materials and reagents used for the experiments. Reagent/ressource Reference or source Identifier or catalog number Chemical, enzymes, materials Accutase Innovative Cell Technologies, Inc. AT-104 ATPlite 1step Luminescence Assay System Revvity Healthy Sciences Inc. Primary antibody anti-ß3 tubulin chicken Abcam ab41489 Secondary antibody anti-chicken coupled Alexa Fluor 488 Abcam ab150173 Blasticidin Invivogen Ant-bl BrdU kit Merck QIA58 BSA Sigma-Aldrich A9647 B-27 Gibco 17504001 Cell scraper Sarstedt 83.3951 CellTracjer Blue CMAC Invitrogen C2110 Culture dishes 10 cm 2 Sarstedt 83.3902.300 Culture dises 20 cm 2 Sarstedt 83.39.03 DAPI Merck 10236276001 DHE Invitrogen D11347 DMEM Sigma-Aldrich D6429 DMSO Sigma-Aldrich 276855 EGTA Thermoscientific 409910250 Fetal bovine serum Corning 35-079-CV Filter 40 µm mesh size PluriSelect 43-57040-01 Filter 10 µm mesh size PluriSelect 43-50010-01 G418 Sigma-Aldrich 108321-42-2 Glutamate Sigma-Aldrich G1626 GlutaMax Thermo Scientific 35050087 HBSS Gibco 14065049 Horse serum BioConcept 2-05F00-1 K-HEPES Research Organics Inc. 6003H KH 2 PO 4 Sigma-Aldrich P5655 Malate Sigma-Aldrich M1000 Mannitol Sigma-Aldrich M4125 MgCl 2 Sigma-Aldrich M4880 MitoSOX Red Invitrogen M36008 MTT Merck M2128 Neurobasal Gibco 21103-049 PBS VWR 392–0440 PFA Sigma-Aldrich P6148 Penicillin/Streptomycin Bioconcept 4-01F00‐5 Retinoic acid Sigma-Aldrich R2625 Saccharose Eurobio 018363 Subtilisin A MedChem Express HY-E70076 Succinate Sigma-Aldrich S2378 TMRM Fluka 87919 Black 96 well plates Greiner 655090 White 96 well plates Greiner 165306 Clear 96 well plates Falcon 353072 Clear 12 well plate Falcon 353043 Collagen I precoated slide Neuvitro GC-18-1.5 Experimental models A172 untransfected ATCC CRL-1620 P301L-Tau-GFP transfected SH-SY5Y neuroblastoma cells (Human) The Götz laboratory Healthy SH-SY5Y neuroblastoma cells transfected with GFP ATCC CRL-2266 Devices and software Eclipse Ti2 Nikon Microscope Nikon Multiplate reader Cytation 3 Agilent Multiplate reader Cytation 5 Agilent Neurites Outgrowth Module Agilent Resipher Lucid Lab Lucid Scientific https://lucidsci.com Take3 Microvolume plate Agilent Fiji (Fiji is just ImageJ) 2.16.0 NIH https://imagej.net/software/fiji/ Prism version 10 GraphPad https://www.graphpad.com/features Huygens Deconvolution Software version 14.10.0 Scientific Volume Imaging https://svi.nl/Huygens-Software Imaris version 9.9.1 Oxford Instruments https://imaris.oxinst.com Cell lines The human neuroblastoma cell line SH-SY5Y (ATCC, CRL-2266) was used as the recipient cells for the mitochondrial transplantation procedure. These cells have been documented in the scientific literature and are widely used as neuronal models in neuroscience research. As a cellular model for tauopathies, we used human neuroblastoma SH-SY5Y cells stably overexpressing the P301L mutation (“P301L cells”) on the MAPT gene of chromosome 17 and tagged with a green fluorescence protein (GFP). This model was developed and generously provided by Professor Jürgen Götz's laboratory at the University of Queensland (Brisbane,Australia) [ 31 ]. The control cells consist in SH-SY5Y cells expressing the GFP-vector only ("vector cells"). For both cell models (vector and P301L cells), 4.5 µg/ml of blasticidin was added to the culture medium to maintain a stable expression of the plasmids. Since intercellular mitochondria transfer naturally occurs between neurons and astrocytes, resulting in enhanced neuronal functions and neuroprotection [ 11 ], a human astrocytic cell line A172 (ATCC, CRL-1620) was used as the mitochondria donor to provide healthy mitochondria for the bioenergetics experiments. In parallel, A172 cells stably expressing the red fluorescent protein (RFP) tagged to mitochondria were used, allowing the visualization of the transplanted astrocytic mitochondria in the neuronal cells for the microscopic experiments. Cell culture SH-SY5Y cells were cultured in a growing medium composed of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% penicillin and streptavidin, 1% glutamax, 10% fetal bovine serum (FBS), and 5% horse serum (HS). The selection antibiotic (blasticidin) was added during the medium change. The cells were cultivated in 10 cm 2 culture dishes containing 10 ml of growing medium. A172 cells were cultured in DMEM supplemented with 1% penicillin/streptavidin, 1% glutamax, and 10% FBS. These cells were maintained in 10 cm 2 culture dishes and replated in a 20 cm 2 before the mitochondrial isolation. The cells were detached from the culture dishes using accutase and split twice weekly once they reached approximately 80% confluency. The cells were subsequently stored in an incubator maintained at 37°C and a CO 2 concentration of 5%. Cellular differentiation To be as close as possible to a neuronal phenotype, SH-SY5Y vector and P301L cells were differentiated into neuronal cells. Cells were plated in a 96-well plate format, and the growing medium was exchanged with 100 µL of differentiation medium 24 hours after the seeding of cells. The differentiation medium comprised Neurobasal medium, 1% penicillin and streptavidin, 2% B-27, and 10 µM retinoic acid. A washing step with phosphate-buffered saline (PBS) was performed before the incubation with the differentiation medium to ensure the complete removal of FBS and HS. The cells were maintained in the differentiation medium for three days. Subsequently, half of the volume of the differentiation medium was replaced to refresh the medium before the mitochondrial transplantation. Mitochondrial isolation The protocol for mitochondrial isolation has been previously established by Preble et al. in 2014 [ 24 ]. All procedures were conducted in a controlled, sterile environment. For each mitochondrial isolation, a full dish of 20 cm 2 of A172 cells was prepared. A homogenization buffer was prepared, composed of 300 mM saccharose, 10 mM K-HEPES, and 1 mM EGTA, which were diluted in ultrapure water. The pH was adjusted to 7.2 with a solution of 1 M NaOH. The solution was filtered under sterile conditions using a 0.2 µm filter and stored at 4°C until use. Medium was removed from the dish containing A172 cells, followed by one wash with PBS. Then, 1 ml of homogenization buffer was added to the dish, and the cells were mechanically harvested using a cell scraper. The cells were centrifuged at 4°C for 5 minutes at 700 x g. Next, the cell pellet was suspended in 2 ml of homogenization buffer, and cells were lysed using a Potter grinder. A solution containing 4 mg/ml Subtilisin A was added to the tube and incubated on ice for 10 minutes. Filtration steps were then performed to remove the majority of cell debris. First, cell lysate was filtered using a 40 µm mesh size filter. Then, 500 µl of 20 mg/ml BSA (bovine serum albumin) was added to the lysate before a second filtration step with a new filter with a 40 µm mesh size, followed by a third filtration with a mesh size of 10 µm. Finally, the filtrated cell lysate was centrifugated at 4°C for 10 minutes at 10,000 x g. The final pellet was suspended in 200 µl of mitochondrial assay solution (MAS) and stored on ice until the transplantation step. Of note, the MAS is composed of 200 mM mannitol, 70 mM saccharose, 10 mM KH 2 PO 4 , 5 mM MgCl 2 , 2 mM K-HEPES, 1 mM K-EGTA, and 0.2% BSA in 200 ml of ultrapure water [ 16 ]. The MAS pH is adjusted to 7.2, and the solution is frozen at -20°C for extended periods. Estimation of mitochondrial concentration The concentration of isolated mitochondria was determined by conducting a protein assay with the Cytation 3 and the Take3 Microvolume plate. The software utilized for data analysis was Gen3.16. The protein absorption was measured at a wavelength of 280 nanometers (nm), and the MAS was employed for the blank measurement. This approach is characterized by its reliability, efficiency, and speed, thereby ensuring optimal conditions for preserving the integrity of isolated mitochondria before transplantation. Mitochondrial transplantation Isolated mitochondria were kept on ice in the MAS for less than 1 hour. The acceptor cells (SH-SY5Y cells) were incubated with the isolated mitochondria (mitochondria-transplanted group), while the MAS alone was used as the control condition. A final volume of 10 µl of isolated mitochondria was added in the 100 µl of medium in 96-wells plate containing neuroblastoma cells. The final concentrations of isolated mitochondria that were tested are 5 µg/ml, 10 µg/ml, 25 µg/ml, and 50 µg/ml. The selection of these concentrations was guided by the findings of Shi et al., who conducted a study to ascertain the most efficacious concentrations for SH-SY5Y cells [ 30 ]. The cell plate was then incubated at 37°C and 5% CO 2 for 24 or 48h. Validation of the entry of isolated mitochondria into recipient cells Vector cells were plated in a 12-well plate on collagen I pre-coated coverslipes at a density of 5 x 10 4 cells/well. Cells were then differentiated by following the differentiation protocol previously described. To investigate whether isolated mitochondria can enter into cells, A172 cells expressing the RFP-tagged mitochondria (mitoRFP) were used as a mitochondrial source. After 24 and 48 hours of co-incubation with isolated mitochondria, Vector cells were incubated with 5 µM of CellTracker Blue dye for 30 minutes at 37°C to visualize the cells’ area. Then, cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature (RT). Pictures were captured using an Eclipse Ti2 widefield microscope (Nikon). Images were subjected to a deconvolution step using the Huygens Deconvolution Software. Subsequent analysis was conducted using the Fiji and Imaris softwares. Cellular viability assay The cellular viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. This colorimetric test reflects the metabolic activity of living cells and is interpreted as an indicator of cellular viability and toxicity. The experiment was conducted by seeding 1.5 x 10 4 SH-SY5Y cells per well in clear 96-well plates. The MTT was first diluted in the culture medium at 5 mg/ml. Then, the diluted MTT was added to the SH-SY5Y cells and incubated for 2 hours at 37°C and 5% CO 2 . The medium was carefully removed from the wells and substituted with 150 µL of dimethyl sulfoxide (DMSO). The plate was then placed on a shaker at 250 rpm for 15 minutes. The resulting absorption was measured at a wavelength of 550 nm using the Cytation 3 Cell Imaging Multi-mode Plate Reader (BioTek, Agilent, Switzerland). This test was conducted 24h and 48h after the mitochondrial transplantation. ATP levels The test was conducted using the "ATPlite 1step Luminescence Assay System" kit. The assay is based on a bioluminescence reaction between an enzyme, luciferase, and a substrate, luciferin. This reaction requires the presence of ATP, with the emitted light being proportional to the level of ATP in each well. The cells were seeded in white 96-well plates (1.5 x 10 4 cells/well) to optimize the signal in the plate reader. An ATP standard (ranging from 0 to 20 µM) was prepared in duplicate to calculate the ATP concentration in our samples. To initiate the enzymatic reaction, 100 µl of ATP substrate was added to each well. Then, the plate was placed on the shaker at 200 rpm for 2 min, and the bioluminescence signal was read with the Cytation 3 instrument. The tests were conducted at 24h and 48h post-transplantation. Determination of Mitochondrial Membrane Potential The mitochondrial membrane potential (MMP) was determined using the TMRM dye (tetramethylrhodamine methyl ester). The dye’s capacity to enter into the mitochondrial matrix is proportional to the MMP. To assess the membrane potential of mitochondria freshly isolated from A172 cells, and to ascertain their bioenergetic activity, two tubes containing 100 µg/ml of isolated mitochondria in MAS were prepared and incubated with 0.4 µM of TMRM for 30 minutes at 37°C. Subsequently, the samples were centrifuged at 10,000 x g for 10 minutes at room temperature. Then, the mitochondria pellet from the first tube was resuspended in the MAS, while the second pellet was resuspended in the MAS supplemented with 0.05 mM Glutamate, 8 mM Succinate, and 5 mM Malate which are substrates required for the electron transport chain activity. Finally, 50 µl of samples were added to black 96-well plates, and the emitted fluorescence (Excitation/Emission: 550/580 nm) was detected using the Cytation 3. To assess the effects of mitochondrial transplantation on the MMP of neuronal cells, SH-SY5Y cells were seeded in black 96-well plates at a density of 1.5 x 10 4 cells/well. The measurement of the MMP was conducted at 24h and 48h following the transplantation with isolated mitochondria. The TMRM dye was added to each well at a final concentration of 0.4 µM. The plate was then placed in the dark on a shaker at 100 rpm for of 30 minutes. Two washing steps were then performed with HBSS, and the fluorescence intensity (Excitation/Emission: 550/580 nm) was measured with the Cytation 3. Assessment of superoxide anion radical levels The total and mitochondrial superoxide anions levels were measured using the DHE (dihydroethidium) and MitoSOX Red dyes, respectively. The cells were plated in black 96-well plates at a density of 1.5 x 10 4 cells/well. The superoxide anion levels were determined at 24h and 48h post-transplantation. The cells were incubated with 10 µM of DHE for 20 minutes in the dark at room temperature or 50 µM of MitoSOX red for 90 minutes at 37°C and 5% CO 2 . Cells were then washed twice with HBSS and the fluorescence intensity (Excitation/Emission: 531/595 nm), which is proportional to the total or mitochondrial superoxide anion levels, was detected with the Cytation 3. Cellular respiration measurement The Resipher device was utilized to assess cellular respiration over time. This device allows the measurement of oxygen consumption rate (OCR) directly in the cell incubator in real time (Lucid Scientific [ 34 ]). The Resipher device was affixed magnetically to a designated lid containing sensors placed on 96-well plates with 1.5 x 10 4 SH-SY5Y cells/well. The medium volume of 90 µl per well was selected based on the recommendations of Lucid lab to ensure optimal measurement conditions. The experimental protocol commenced 24h prior to transplantation and concluded 48h post-transplantation. The OCR values were extracted on the Lucid Scientific platform. BrdU assay To ascertain that the observed effect after the mitochondrial transplantation is not attributable to a proliferative effect, a BrdU (Bromodeoxyuridine) kit was used. In brief, the BrdU (1:2000) was incubated with undifferentiated SH-SY5Y cells overnight to allow one complete cycle of the cell division process. The following day, the sample underwent fixation and denaturation, followed by anti-BrdU immunostaining. The anti-BrdU secondary antibody is coupled with the horseradish peroxidase (HRP) enzyme, producing a colored product. The reaction is then halted using a solution provided in the kit. The absorbances were measured at 450 nm and 540 nm using the Cytation 3. The final values correspond to the difference between the absorbances at 450 nm and 540 nm. Neurite outgrowth measurement The impact of mitochondrial transplantation on neuronal plasticity was assessed using the Agilent BioTek Gen5 neurite outgrowth module. Cells were seeded in black 96-well plates and maintained in the differentiation medium for three days. The differentiation medium comprised Neurobasal, 1% penicillin, 1% streptavidin, 2% B27, and 10 µM retinoic acid. Thereafter, the differentiation medium was replaced with Neurobasal medium containing 1% penicillin and streptavidin, 2% B27, and different treatment conditions: MAS only (control), isolated mitochondria, or 50 ng/ml nerve growth factor (NGF) as a positive control. After 48h of treatment, an immunostaining with anti-ß3 tubulin was performed. Cells were fixed with 4% PFA for 10 minutes at RT and permeabilized with 0.15% Triton in PBS for 15 minutes at room RT. Samples were incubated with 2% BSA in PBS to block unspecific sites for 1 hour. The primary antibody (anti-ß3 tubulin, chicken, 1:1000) was incubated overnight at 4°C. The secondary antibody (anti-chicken and preabsorbed with Alexa Fluor 488, 1:500) was subsequently incubated for 1 hour in the dark at RT. Finally, the cells were incubated with a nuclear staining solution (DAPI, 1:1000) for 5 minutes at RT. The samples were then preserved in PBS and stored at 4°C. Data from the neurite outgrowth experiments were obtained with the Cytation 5 Cell Imaging Multi-mode Plate Reader (BioTek, Agilent, Switzerland). Pictures were submitted to a preprocessing step with the predefined parameters in the software for both channel DAPI and GFP (rolling ball diameter: 138 µm). Then, a deconvolution step was applied to the preprocessed images (Point spread function: “Auto, based on objective”; iterations number: 5). The neurite outgrowth module was applied to the deconvolved images with experimental setting established for vector and P301L cells. The parameters collected for this study were: the average neurite length (the total skeleton length of all neurites divided by the soma count), the average neurite count per cell (number of neurites connected to the identified soma), average neurites branches per cell (count the number of ending branches connecting to neurites divided by the soma count) and the neurite thickness ( correspond to the total neurite area divided by the total neurite length, in µm). Statistical analysis All results were exported to Microsoft Excel 2019® and analyzed using GraphPad Prism 10® software. The results, depicted as boxes and whisker plots, indicate the median value, the mean, and the distribution of values with the minimal and maximal points ("whiskers"). The values are expressed as percentages of the control (MAS) condition. To ensure the reliability and reproducibility of the experimental results, each experiment was performed at least three times. The statistical analysis employed was One-way ANOVA with a post-hoc Dunnett's multiple comparison test versus the control condition or Two-ways ANOVA with a post-hoc Tuckey's multiple comparison test. Results were considered statistically significant if p-values < 0.05. Results 1. Establishment of the optimal mitochondrial transplantation parameters on the SH-SY5Y cellular model. Astrocytic mitochondria were freshly isolated from the A172 cells. Microscopic experiments were performed to assess the ability of isolated mitochondria to enter into recipient cells. As shown in the microscopy pictures and 3D representations in Fig. 1 a, the isolated mitochondria are present in the recipient cells after 24h and 48h of incubation (Supplementary Fig. 1). To ensure that the isolated mitochondria were still healthy and functional, we first assessed whether their mitochondrial membrane potential (MMP) was preserved and the electron transport chain (ETC) activity was still efficient. We observed that the TMRM dye was still capable of entering into isolated mitochondria and emitting a fluorescent signal, which reflected a polarized membrane potential (Fig. 1 b). The addition of ETC substrates (glutamate, succinate, and malate) resulted in an augmentation of MMP in comparison without substrates. These results indicated that the structural and functional integrity of mitochondria was preserved after the isolation process. Next, we determined the optimal concentration and incubation time for the transplantation of isolated mitochondria. Initial trials were conducted on healthy and undifferentiated SH-SY5Y cells. Based on the methodologies outlined by Shi et al. [ 30 ], a range of concentrations were selected for subsequent experimentation: 5, 10, 25, and 50 µg/ml, and two time points: 24h and 48h. First, we assessed whether the treatment with isolated mitochondria was toxic to cells. To do so, we performed an MTT viability assay. No significant difference was observed between the control (no mitochondria) and all the tested conditions (from 5 µg/ml to 50 µg/ml) at 24h after mitochondria transplantation (Fig. 1 c). Although mitochondrial transplantation did not increase cell viability, it was not toxic for the cells. In parallel, we checked the effect of the treatment on the metabolic activity of the cells using an ATP assay. The same experimental conditions were used previously for the MTT assay. A dose-response curve was observed after one day of transplantation, with a significant increase for the treated conditions compared to the control group (Fig. 1 d). Especially, a + 9% and + 19% increase in ATP levels were observed with the 25 and 50 µg/ml, respectively. These two treatment concentrations were selected to continue the screening phase. We confirmed the MTT and ATP results 48h after transplantation. As observed on the graphs (Fig. 1 e, Fig. 1 f), the same trend was observed after two days of treatment, with no difference between the control and treated conditions regarding the cellular viability (Fig. 1 e) but an increase in cellular metabolic activity (+ 10% and + 12% increase for the 25 and 50 µg/ml conditions, respectively) (Fig. 1 f). Since it is known that ATP production is closely related to the MMP, this parameter was measured using the TMRM dye. In comparison with the control group, no difference was observed after 24h of treatment (Fig. 1 g). A significant increase in MMP was detected after 2 days of incubation for both treated conditions compared to the control (Fig. 1 h). At this time point, MMP is statistically higher in the 50 µg/ml condition (+ 96% increase versus the control) than in the 25 µg/ml condition (+ 48% increase versus the control). A BrdU assay was performed to ensure that these beneficial effects were not due to an increase in cell proliferation after treatment. No variation in cell proliferation was observed at 24h post-transplantation for both tested concentrations (Fig. 1 i). In addition, a decrease in this parameter (− 9%) was observed after treatment with 25 µg/ml of isolated mitochondria for 48h (Fig. 1 j). Therefore, all the beneficial effects observed in the previous data were due to mitochondrial transplantation and not to cell proliferation. Based on all these results, the 25 µg/ml mitochondria concentration was selected for subsequent analysis. Indeed, this concentration showed a beneficial effect on cell functions with no obvious difference with the 50 µg/ml treatment condition. 2. Isolated mitochondria improve bioenergetic functions and reduce the oxidative stress of the vector SH-SY5Y cells. To get closer to a neuronal model, the second part of the experiments was performed on differentiated and healthy SH-SY5Y cells ("vector" cells). Based on the previous results from the screening phase, the optimal treatment concentration was 25 µg/ml. The same bioenergetic assays were performed on this cell model, with, in addition, the assessment of superoxide anion radical levels and cellular respiration at 24h and 48h post-transplantation. The potential toxicity of the treatment was assessed at 24h and 48h post-transplantation. We observed an increase in viability compared to the corresponding control condition of + 28% and + 34%, respectively. However, no difference was observed between the treated condition at 24h and 48h (Fig. 2 a). An increase in cellular metabolism was also observed, with + 25% and + 45% increase in ATP production after treatment at 24h and 48h, respectively. For this parameter, the production of ATP is significantly higher at 48h post-transplantation compared to the treated group at 24h (Fig. 2 b). In parallel, MMP was increased of + 24% and + 31% after one and two days of treatment, respectively, showing a correlation with ATP levels. However, there was no difference between the treated condition at 24h and 48h after transplantation (Fig. 2 c). We next assessed whether the treatment's effects were related to an increase in cellular respiration by measuring the oxygen consumption rate (OCR) in the recipient cells. Compared to the corresponding control condition, an increase in OCR was observed at 24h (+ 42%) and 48h (+ 52%) after transplantation. This increase appeared stable over time, as there was no significant difference between the treated conditions at both time points (Fig. 2 d). The main risk in improving the bioenergetic functions of cells is the parallel increase in the production of reactive oxygen species (ROS). Therefore, cellular and mitochondrial superoxide anion radical levels were measured using fluorescent dyes. Surprisingly, mitochondrial superoxide anion levels were decreased at 24h (-14%) and 48h (-16%) after treatment. No difference was observed between the two time-points (Fig. 2 e). Therefore, this effect is maintained over time. In addition, total superoxide anion radical levels were decreased at 48h (-12%) after transplantation with isolated mitochondria (Fig. 2 f). Thus, mitochondrial transplantation improves the cellular viability and bioenergetic functions of differentiated vector cells. In addition, a decrease in oxidative stress was observed after treatment. Most of the results appeared at 24h and 48h post-transplantation, which means that the transplantation might have a positive effect in the long term. 3. Mitochondrial transplantation improves the bioenergetics functions in P301L SH-SY5Y cells and reduces the mitochondrial superoxide anions levels. Since the results of the tests with the control (vector) cells were maintained at 24h and 48h, the experiments with the P301L SH-SY5Y cells were performed at the same time points. The cellular deficits of the P301L model compared to the control cells have been previously demonstrated in our laboratory [ 12 ]. Cell viability was improved at 48h post-transplantation with an increase of + 22% compared to the untreated condition. No significant differences were observed at 24h (Fig. 3 a). In parallel, an increase in ATP production was observed at 24h (+ 17%) and 48h (+ 31%) compared to the control condition. This time, the effect of the treatment was higher at 48h than 24h (Fig. 3 b). The MMP was significantly increased in P301L 24h post-transplantation, however this effect was lost after 48 hrs (Fig. 3 c). Cellular respiration was also examined in P301L cells. An increased OCR was observed after treatment at 24h (+ 18%) and at 48h (+ 36%) compared to the corresponding control condition (Fig. 3 d). As for the ATP production, the respiratory capacity of the cells seemed to correlate with the incubation time of the isolated mitochondria with the P301L cells. A previous study of our group has shown that ROS level is higher in the P301L cells than in the control cells [ 12 ]. Thus, the impact of mitochondrial transplantation on oxidative stress is an essential question in this case. Therefore, total and mitochondrial superoxide anion radical levels were assessed in P301L cells after mitochondria transplantation. We observed a significant decrease in mitochondrial ROS levels at 24h (-13%) and at 48h (-9%) compared to the corresponding control condition (Fig. 3 e). This effect appeared to be stable over time, with no statistical difference between the treated condition at either time point. However, no decrease in total ROS levels was observed after treatment (Fig. 3 f). Overall, mitochondrial transplantation improved the cellular viability and bioenergetic functions of P301L cells after 24h and 48h of incubation. In addition, mitochondrial oxidative stress is reduced after treatment. 4. Mitochondrial transplantation increases the neurite outgrowth in the vector and P301L cells. We showed that mitochondrial transplantation improved the bioenergetic characteristics of the vector and P301L after 48h of incubation. Because mitochondrial bioenergetics is essential in building neuronal networks by providing the energy necessary for neurite formation, a morphological analysis of neurite outgrowth was performed on the vector and P301L cells after two days of treatment with mitochondria. The characterization of the neurite outgrowth parameters of the P301L cells in comparison with the vector is shown in the supplementary Fig. 2. In these experiments, nerve growth factor (NGF) was used as a positive control, and all the neurite outgrowth parameters were measured after 48h of treatment. To measure several parameters characterizing neurite outgrowth, we used the Cytation 5 (Agilent) and the "Neurite Outgrowth Module", which allowed us to generate masks highlighting the soma and neurites of the neuronal cells (Fig. 4 a, Fig. 4 f). Neurite outgrowth parameters were automatically calculated by the Gen5 imaging software. The vector and P01L cells treated with the positive control NGF presented an increase in the average neurite count (Fig. 4 b, Fig. 4 g), the average neurite branches (Fig. 4 c, Fig. 4 h), the average neurite length (Fig. 4 d, Fig. 4 i) and a decrease in neurite thickness (Fig. 4 e, Fig. 4 j), indicating an enhanced neuronal differentiation and plasticity. In the mitochondria-treated cell, we observed an increase in the average number of neurites and branches per cell in the vector (+ 30% and + 19%, respectively) (Fig. 4 b, Fig. 4 c) and P301L cells (+ 51% and + 50%, respectively) compared to the control condition (Fig. 4 g, Fig. 4 h). In addition, neurite length was also increased after treatment for P301L cells (+ 42%) (Fig. 4 i). Of note, the average neurite length observed after mitochondrial transplantation in P301L cells was as high as with the NGF treatment. In parallel, a decrease in neurite thickness was observed for both cell lines (-9% and − 13% for vector and P301L cells, respectively) (Fig. 4 e, Fig. 4 j). In conclusion, mitochondrial transplantation positively impacted the neuronal differentiation process, especially in P301L, which presented an increase in every neurite outgrowth parameter. Discussion In this study, we hypothesized that mitochondrial transplantation could improve the cellular viability, bioenergetic functions, and neurite outgrowth of healthy and tau mutant P301L cells. In the first step, we optimized the mitochondria isolation protocol and determined the optimal treatment concentration. Our data indicate that the protocol used for the mitochondrial isolation preserved mitochondrial function and integrity because the mitochondria were still bioenergetically active after the process. Moreover, we showed that the isolated mitochondria can enter the recipient cells. In line with that, a previous study from Shi et al., have shown that isolated mitochondria can enter the recipient cells after only 30 min of incubation [ 30 ]. In our study, we observed that isolated mitochondria were present in the recipient cells after 24h and 48h of incubation. In the next step, we showed that the mitochondrial transplantation was not toxic for the neuronal cells, and that it had positive impacts on the respiratory chain activity by increasing the ATP and MMP levels. The BrdU assay revealed that the beneficial effects observed were not due to a proliferative effect of the treatment. Moreover, the decrease in cell proliferation observed after 48h of treatment in undifferentiated SH-SY5Y cells might be due to modulatory effects of the mitochondrial transplantation on neuronal differentiation. Indeed, the promoting effect of mitochondrial transplantation on neuronal differentiation was previously shown on schizophrenia-derived induced pluripotent stem cells (iPSCs) differentiated into glutamatergic neurons [ 27 ]. These data are in line with the results obtained in our neurite outgrowth experiments. After this screening phase, we selected the 25 µg/ml mitochondria concentration for further experiments on differentiated SH-SY5Y cells. Because undifferentiated SH-SY5Y cells are not representative of mature neurons, the cells were differentiated to get as close as possible to neuronal cells [ 8 ]. Mitochondria transplantation was performed after differentiation of the vector and the P301L SH-SY5Y cells. The cellular viability, ATP production, MMP levels, and cellular respiration were improved at 24h and 48h after the treatment for the vector cells. The effects of mitochondrial transplantation on the bioenergetic functions were more potent in the differentiated cells than the undifferentiated ones. One explanation could be that the bioenergetic metabolism of undifferentiated and differentiated cells is not the same. Indeed, the undifferentiated cells have a more glycolytic metabolism, while the differentiated cells primarily use the OXPHOS process [ 29 ]. So, it might be possible that mitochondrial transplantation boosts the respiratory activity of the differentiated cells, with beneficial effects on the OCR, ATP production and MMP. In addition, a decrease in the total and mitochondrial superoxide anions levels was observed after the mitochondrial transplantation, in line with the increased cellular viability. These positive effects of the transplantation are reproducible between the 24h and the 48h timepoint, suggesting that treatment might be effective on the long-term. Longer treatment durations will have to be tested in future experiments to determine how long the effects of the transplantation are lasting. The second main aim of this study was to determine whether mitochondria transplantation could improve bioenergetic parameters in a cellular model of tauopathies overexpressing the P301L-tau mutation in the MAPT gene [ 35 ]. This model is mainly associated with frontotemporal dementia but is also widely used as a general model of tauopathies. In particular, the P301L-tau mutation is associated with abnormal hyperphosphorylation of the tau protein with neuronal, synaptic, and mitochondrial dysfunctions. The P301L-tau overexpressing SH-SY5Y cells have been previously characterized in our laboratory, and their bioenergetic deficits in comparison with the vector cells have been demonstrated [ 11 , 15 ]. Based on these previous findings, this study aimed to determine whether mitochondrial transplantation could improve bioenergetic functions in the P301L model. We observed increased cellular viability, ATP levels, MMP, and OCR after treatment with mitochondria. In addition, the positive effects were maintained for at least 48h. Regarding the MMP, we observed that the mitochondria transplantation effect was lost in the P301L cells after 48h, as indicated by a decrease in the fluorescent signal compared to the 24h-treated group. Our hypothesis to explain this result is that a negative feedback loop might be activated in response to the high increase in ATP production after treatment. Indeed, the individual activity of each mitochondrion (the endogenous and the transplanted one) might possibly be lower with a MMP similar to the control condition. However, the global bioenergetic activity is higher than the control condition because mitochondria were added to the system, resulting in higher mitochondrial mass. This regulation can also play an “antioxidant role” in limiting the ROS production and oxidative stress in cells. On top of that, the transplanted mitochondria may have an antioxidant effect by decreasing the mitochondrial superoxide anion levels. It is known that the P301L cells have a higher level of ROS than the vector cells. Therefore, the healthy transplanted mitochondria may bring their own antioxidant system, including the manganese superoxide dismutase (Mn-SOD), which is highly present in the mitochondrial matrix [ 33 ]. It has also been shown that the glutathione reductase levels were increased after the mitochondrial transplantation, suggesting that this enzyme might enhance the antioxidant system in the P301L cells [ 30 ]. Most of the bioenergetic properties of vector and P301L cells have been positively affected by mitochondrial transplantation. Therefore, it was interesting to focus also on morphological characteristics of the treated cells, such as the neurite outgrowth. It has been demonstrated in a hybrid model of mouse neuroblastoma and rat glioma NG108-15 overexpressing the cDNA of human tau441, that tau cells have a higher number of neurites, and these neurites were thicker than in the wild-type cells [ 38 ]. In line with that, our P301L-tau overexpressing cells present shorter neurites but more neurites per cell and more neurite branches than the vector cells. Indeed, the hyperphosphorylated tau protein is known to destabilize the microtubules, which are involved in the elongation of neurites. The higher number of neurites and branches in the P301L could be a way to compensate for the deficit in neurite length. It has been shown in a previous study that mitochondrial transplantation can enhance the neurite growth in vitro on 6-OHDA-induced PC12 cells as a model of Parkinson's disease [ 4 ]. Our results are in line with these findings, as we observe an increase in the average number of neurites and branches per cell for the vector and P301L cells. The neurites length was also enhanced in the P301L cells, which suggests that the mitochondrial transplantation improves the neurites elongation capacity of these cells. Therefore, mitochondrial transplantation improved bioenergetics functions and neuroplasticity process in the context of tauopathies, making this therapeutical approach relevant in tau-related neurodegenerative disorders. Based on the pivotal role of mitochondria in neurons, this approach is now increasingly used in brain disorders studies, such as ischemia, Parkinson’s disease, and traumatic brain injury [ 30 , 36 , 39 ]. Several studies have shown improvement in bioenergetics functions, neuroprotective effects, neurogenesis, and anti-inflammatory response after mitochondrial transplantation in different in vivo and in vitro models [ 26 ]. Indeed, it has been demonstrated that mitochondrial transplantation improves the general mitochondria activity in cells with an increase in ATP production and a decrease in neuronal loss, a decreased inflammation in the hippocampus, and a reduction in ROS levels in a mouse model of AD induced by the injection of ß-amyloid in the intracerebroventricular site or the hippocampus [ 22 , 37 ]. In addition, in the same AD animal model, an increase in memory capacities and restoration of cognitive deficits have been shown after transplantation. In line, mitochondrial transplantation induced a decrease in the “depressive-like” behavior on a lipopolysaccharide-induced model of depression [ 35 ]. Improvement in the locomotor activity has also been observed after mitochondrial transplantation in a Parkinson’s disease mice model [ 4 ]. These effects were explained by the capacity of the transplanted mitochondria to module the gene expression in the recipient cells. Indeed, a few hours after the transplantation, genes related to mitochondrial function and neuronal repairment were upregulated, while the genes involved in the oxidative stress and apoptosis were downregulated [ 37 ]. The sirtuin 1 (SIRT1) pathway appeared to be recruited to ensure the positive effects observed by promoting BDNF production and autophagy in both in vitro and in vivo models [ 26 ]. However, investigations on the underlying mechanisms and specific pathways recruited for the entry and the fate of mitochondria in the recipient cells are still missing [ 19 ]. Mitochondrial transplantation has been more studied in the context of cardiac disease, where the first clinical study on pediatric patients has been realized [ 10 , 23 ]. These patients presented cardiac ischemia following a cardiac surgery event. Autologous mitochondria were isolated from the patient's accessible skeletal muscle, such as the rectus abdominis and the pectoralis, and were delivered by direct injection in the ischemic cardiac areas [ 9 ]. The authors did not observe adverse side effects in the short-term (including arrhythmia or scarring complications); four patients out of five had improved their ventricular function and no longer needed extracorporeal membrane oxygenation (ECMO) support. Therefore, this first clinical study has shown that this therapeutic strategy is promising for several diseases. The first clinical trial in the context of brain ischemia is currently underway (ClinicalTrials.gov: NCT04998357). As for the clinical trial in cardiac disease, this project aims to test the potential of autologous mitochondrial transplantation. The objective is to investigate whether the treatment could reduce the infarct volume. Mitochondria will be isolated from the muscle tissue close to the surgical access site. The treatment should be delivered in the brain artery via a micro-catheter during reperfusion. The presence or absence of side effects will be verified following the clinical trial scheduled for late 2026. In these two clinical trials, the source of mitochondria was the muscle of the patient itself. In our study, the A172 cells were chosen as mitochondrial sources because it has been shown in vitro that astrocytes can naturally transfer their mitochondria to neurons under stress conditions [ 11 , 13 ]. In addition, the most relevant sources of mitochondria would be the same tissue as the targeted organ [ 20 ]. Therefore, the source of mitochondria to ensure the optimal effects of the transplantation is a relevant question, considering that their activity depends on their tissue origin [ 4 ]. Moreover, the delivery way of mitochondria is also a crucial part of the treatment. Indeed, a simple co-incubation, as realized in our study, is not feasible in a clinical context. Although, the intracerebral injection looked efficient in some studies on animal models, it might be too invasive to be applicable on human. Other studies used the intravenous injection, and they found the isolated mitochondria in different organs, including the brain, suggesting that isolated mitochondria can cross the blood-brain barrier [ 21 , 22 ]. The method of mitochondria delivery should be more specific to target the brain specifically. The nasal route represents an interesting option because it is non-invasive and does not require a high amount of mitochondria[ 2 , 3 , 26 ]. To our knowledge, no previous study has focused on the effects of mitochondrial transplantation in tauopathies. Our results have shown that mitochondrial transplantation improves bioenergetic functions and neurite outgrowth in a neuronal model of tauopathies. Therefore, further studies on the impact of mitochondrial transplantation on the other pathological features of tauopathies are needed. Deciphering the impact of mitochondrial transplantation on tau hyperphosphorylation, abnormal tau accumulation in neuronal cells, as well as on mitophagy and microtubule stabilization processes, might provide new insights supporting its potential as a therapeutic strategy for tauopathies. Further studies should be performed to verify our results on more advanced cellular models, like human iPSCs -derived neurons, and in animal models of tauopathy, considering all the preceding questions for clinical application. To conclude, mitochondrial transplantation represents a promising therapeutic approach to boost neuronal bioenergetics and alleviate abnormal tau-related neuronal impairments. In this approach, mitochondria are not a target for therapeutic intervention but represent the therapy themselves. Abbreviations ATP: adenosine triphosphate BrdU: bromodeoxyuridine BSA: bovine serum albumin DHE: dihydroethidium DMEM: Dulbecco’s modified Eagle medium DMSO: dimethyl sulfoxide FBS: fetal bovine serum GFP: green fluorescent protein HRP: horseradish peroxidase HS: horse serum iPSCs: induced pluripotent stem cells MAPT: microtubule associated protein Tau MAS: mitochondrial assay solution MMP: mitochondrial membrane potential MT: mitochondrial transplantation MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NGF: nerve growth factor OCR: oxygen consumption rate PBS: phosphate buffered saline PFA: paraformaldehyde RFP: red fluorescent protein ROS: reactive oxygen species RPM: revolutions per minute RT: room temperature TMRM: tetramethylrhodamine methyl ester Declarations Acknowledgement This work was supported by grants from the OPO Foundation, the Novartis Forschungsstiftung FreeNovation and the Dementia Research, Synapsis Foundation Switzerland (2022-PI05) and the Bürgenstock Foundation. We are grateful to Olivier Buetzberger from the Agilent company for his help and availability during the installation and training of the neurite outgrowth module. We thank Fides Meier for the technical support. Fundings This work was supported by grants from the OPO Foundation, the Novartis Forschungsstiftung FreeNovation and the Dementia Research, Synapsis Foundation Switzerland (2022-PI05) and the Bürgenstock Foundation. Competing Interests The authors declare they have no competing interests. Author contributions Conceptualization: A.G.; Methodology: AB, AR; Data collection: A.B., A.G., A.R.; Formal analysis: A.B., A.G., A.R.; writing-original draft: A.B., A.G.; Funding acquisition: A.G.; Resources: A.G., A.E., Supervision: A.G., A.E., A.P. All authors have read and agreed to the published version of the manuscript. Data availability The corresponding data presented in this study are available on Open Science Framework (https://osf.io/2pju6/?view_only=6f3983caf10546e881dbd2fa6cb70a98). Clinical trial number Not applicable References Agnello L, Ciaccio M (2022) Neurodegenerative Diseases: From Molecular Basis to Therapy. Int J Mol Sci 23 Alexander JF, Mahalingam R, Seua AV et al. (2022) Targeting the Meningeal Compartment to Resolve Chemobrain and Neuropathy via Nasal Delivery of Functionalized Mitochondria. Advanced Healthcare Materials 11 Chang J-C, Chao Y-C, Chang H-S et al. (2021) Intranasal delivery of mitochondria for treatment of Parkinson’s Disease model rats lesioned with 6-hydroxydopamine. Scientific Reports 11 Chang J-C, Wu S-L, Liu K-H et al. (2016) Allogeneic/xenogeneic transplantation of peptide-labeled mitochondria in Parkinson's disease: restoration of mitochondria functions and attenuation of 6-hydroxydopamine–induced neurotoxicity. 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Supplementary Files Supplementaryfigure.docx Supp.Fig.1aMitochondria24h.mp4 Supp.Fig.1bMitochondria48h.mp4 Cite Share Download PDF Status: Published Journal Publication published 10 Dec, 2025 Read the published version in Molecular Neurobiology → Version 1 posted Editorial decision: Revision requested 29 May, 2025 Reviews received at journal 28 May, 2025 Reviewers agreed at journal 23 May, 2025 Reviews received at journal 19 May, 2025 Reviewers agreed at journal 18 May, 2025 Reviewers agreed at journal 18 May, 2025 Reviewers invited by journal 17 May, 2025 Editor assigned by journal 09 May, 2025 Submission checks completed at journal 09 May, 2025 First submitted to journal 22 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6502413","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":450531073,"identity":"b80bad09-3c33-4c79-93fa-b5ce6b0a014d","order_by":0,"name":"Aline Broeglin","email":"","orcid":"","institution":"University of Basel","correspondingAuthor":false,"prefix":"","firstName":"Aline","middleName":"","lastName":"Broeglin","suffix":""},{"id":450531074,"identity":"5d95a4f6-e9e3-4c2e-93cf-2aae490ba2d7","order_by":1,"name":"Aurélien Riou","email":"","orcid":"","institution":"University of Basel","correspondingAuthor":false,"prefix":"","firstName":"Aurélien","middleName":"","lastName":"Riou","suffix":""},{"id":450531075,"identity":"39f38bc3-b6bd-4d1a-980f-0ca9d7c4325c","order_by":2,"name":"Andreas Papassotiropoulos","email":"","orcid":"","institution":"University of Basel","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Papassotiropoulos","suffix":""},{"id":450531076,"identity":"34e671c7-4b62-4be7-a358-7d21dfa10367","order_by":3,"name":"Anne Eckert","email":"","orcid":"","institution":"University Psychiatric Clinics (UPK)","correspondingAuthor":false,"prefix":"","firstName":"Anne","middleName":"","lastName":"Eckert","suffix":""},{"id":450531077,"identity":"df599c81-fc2c-4713-b322-d3bc462362c3","order_by":4,"name":"Amandine Grimm","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYLACHgMIfYCBwYaBgZkHzEkgVksasVoQzMNwLk4t/NMOP3vwpuCOHAP/2oeHbuacT9zOznvw4w8GuzxcWiRup5kbzjF4Zswg8dzgcO6224k7m/mSpXkYkotxOup2gpk0j8HhxAaJYwxgLRsO8xhIA/2V2IBDh/zt9G8gLfVQLedAWox//sCjxeB2DtiWBAb+NpCWAyAtZhI8eLQY3s4pk5xjcNiwTYINpCXZeMNhvjRrHoNknFrkbqdvk3jz57A8P/8x5s+52+xkN5w/e/jmjwo7nFrggE0iAcXBhNSDAP8BYlSNglEwCkbBSAQAE4RbVtcM9cwAAAAASUVORK5CYII=","orcid":"","institution":"University of Basel","correspondingAuthor":true,"prefix":"","firstName":"Amandine","middleName":"","lastName":"Grimm","suffix":""}],"badges":[],"createdAt":"2025-04-22 09:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6502413/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6502413/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12035-025-05604-y","type":"published","date":"2025-12-10T15:58:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81828564,"identity":"9fb0362b-9151-4ab6-8746-a19602d8e879","added_by":"auto","created_at":"2025-05-02 13:10:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":155276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial transplantation in undifferentiated SH-SY5Y cells. (a) \u003c/strong\u003e\u003cem\u003eControl (Vector) SH-SY5Y cells stained with the CellTracker blue dye (green false color) , and mitochondria isolated from A172 cells expressing the MitoRFP tag are shown in red. Images were captured at 100x magnification with the fluorescent Eclipse Nikon Microscope after 24h and 48h of incubation with isolated mitochondria. 3D representations were obtained with the Imaris software; the white arrows shows to transplanted mitochondria into the recipient cells. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Determination of MMP of isolated mitochondria. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec, e\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Estimation of cellular viability at 24h (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and 48h (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) post-transplantation with 5 µg/ml, 10 µg/ml, 25 µg/ml, and 50 µg/ml of isolated mitochondria. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed, f\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Relative quantification of ATP levels at 24h (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and 48h (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e). (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eg-h\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e)Determination of MMP at 24h (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eg\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and 48h (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eh\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) after mitochondria transplantation. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ei-j\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Cellular proliferation effects of mitochondrial transplantation at 24h (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ei\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and 48h (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ej\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e). The boxes represent the median (full line) and the mean (“ + ” symbol), and the whiskers represent the minimal and maximal values. Each data set represents N=3 independent experiments (n= 20-30 replicates per condition). Values are shown as the percentage of the control condition. One-way ANOVA and post hoc \u003c/em\u003eDunnett's multiple comparison test versus the control condition\u003cem\u003e. *p\u0026lt; 0,05 ; **p\u0026lt;0,01 ; ***p\u0026lt;0,001. ATP: adenosine triphosphate, MMP: mitochondrial membrane potential.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6502413/v1/49563f69ede50c4212f864a6.png"},{"id":81828562,"identity":"18e89521-a8ac-45c5-961f-097604687b58","added_by":"auto","created_at":"2025-05-02 13:10:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of mitochondrial transplantation on the cellular viability and bioenergetic parameters in differentiated vector cells. \u003c/strong\u003eThe « control » condition corresponds to the vehicle-treated condition with the mitochondrial assay solution. The « mitochondria » condition corresponds to the treatment with 25 µg/ml of isolated mitochondria. \u003cem\u003e(\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Estimation of cellular viability at 24h and 48h post-transplantation. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Relative quantification of ATP levels at 24h and 48h post-transplantation. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Determination of MMP at 24h and 48h post-transplantation. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Measurement of OCR at 24h and 48h post-transplantation. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Relative levels of mitochondrial superoxide anions at 24h and 48h post-transplantation. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Relative quantification of the total superoxide anions at 24h and 48h post-transplantation (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eg\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e). The boxes represent the median (full line) and the mean (“ + ” symbol), the whiskers represent the minimal and maximal values. Each data represent N=3 independent experiments (n= 30-40 replicates per condition). Values are shown as the percentage of the control condition. Two-way ANOVA and post hoc Tukey’s multiple comparison tests. *p\u0026lt; 0,05 ; **p\u0026lt;0,01 ; ***p\u0026lt;0,001. ATP: adenosine triphosphate, MMP: mitochondrial membrane potential, OCR: oxygen consumption rate.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6502413/v1/79d2308fcbee7c645cc87124.png"},{"id":81828957,"identity":"64eab156-2819-4acf-afed-493b5ad13d60","added_by":"auto","created_at":"2025-05-02 13:18:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54756,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of mitochondrial transplantation on the cellular viability and the bioenergetic features in differentiated P301L cells. \u003c/strong\u003eThe « control » condition corresponds to the vehicle-treated condition with the mitochondrial assay solution. The « mitochondria » condition corresponds to the treatment with 25 µg/ml of isolated mitochondria. \u003cem\u003e(\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Estimation of cellular viability at 24h and 48h post-transplantation. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Relative quantification of ATP levels at 24h and 48h post-transplantation. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Determination of MMP at 24h and 48h post-transplantation. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Measurement of OCR at 24h and 48h post-transplantation. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Relative levels of mitochondrial superoxide anions at 24h and 48h post-transplantation. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Relative quantification of the total superoxide anions at 24h and 48h post-transplantation (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eg\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e). The boxes represent the median (full line) and the mean (“ + ” symbol), the whiskers represent the minimal and maximal values. Each data represent N=3 independent experiments (n= 30-40 replicates per condition). Values are shown as the percentage of the control condition. Two-way ANOVA and post hoc Tukey’s multiple comparison tests. *p\u0026lt; 0,05 ; **p\u0026lt;0,01 ; ***p\u0026lt;0,001. ATP: adenosine triphosphate, MMP: mitochondrial membrane potential, OCR: oxygen consumption rate.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6502413/v1/ec9ab635637d63670c9fae29.png"},{"id":81828566,"identity":"965dcbf5-49bd-40af-be1f-7cafe1801596","added_by":"auto","created_at":"2025-05-02 13:10:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":401270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpcat of mitochondrial transplantation on neurites outgrowth parameters in the vector and the P301L cells. \u003c/strong\u003eThe « control » condition corresponds to the vehicle-treated condition with the mitochondrial assay solution. The « mitochondria » condition corresponds to treatment with 25 µg/ml of isolated mitochondria. The « NGF » condition corresponds to the positive control in which cells were treated with 50 ng/ml of NGF. \u003cstrong\u003e(a, f) \u003c/strong\u003e\u003cem\u003eCells were stained for ß3 tubulin (green color) and DAPI (blue color). All the pictures were captured 48h post-transplantation at 20x magnification. Pictures of vector (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and P301L (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) cells were acquired with the Cytation 5, and the neurites analysis was performed with the neurites outgrowth module from Agilent. (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb, g)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Average neurites count per cell in vector (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and P301L cells (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eg\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e). (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec, h\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Average neurites branches in vector (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and P301L cells (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eh\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e).(\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed, i\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Average neurites length in vector (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and P301L cells (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ei\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e). (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ee, j\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Neurites thickness of vector (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) and P301L (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003ej\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) cells. The boxes represent the median (full line) and the mean (“ + ” symbol), the whiskers represent the minimal and maximal values. Each data set represents N=3 independent experiments (n= 40-50 images analyzed per condition). Values are shown as the percentage of the control condition. One-way ANOVA and post hoc Tukey’s multiple comparison tests. *p\u0026lt; 0,05 ; **p\u0026lt;0,01 ; ***p\u0026lt;0,001. NGF: nerve growth factor.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6502413/v1/8ad1da200676181a4fc46a3d.png"},{"id":98244629,"identity":"bf8e899e-4dfa-4e06-8432-45309386f6d3","added_by":"auto","created_at":"2025-12-15 16:14:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2041708,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6502413/v1/6fabb1bf-af03-4b2d-82af-b4c1fae1816b.pdf"},{"id":81828958,"identity":"e0b47933-e7da-4269-a369-cc5cc569cd45","added_by":"auto","created_at":"2025-05-02 13:18:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":318934,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-6502413/v1/539825a3a4138e0b8003d575.docx"},{"id":81828580,"identity":"0c5b335e-4a2b-4eed-9b1a-17638eca26c2","added_by":"auto","created_at":"2025-05-02 13:10:05","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2849510,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.Fig.1aMitochondria24h.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6502413/v1/9833b0e52d118f4affaf9c92.mp4"},{"id":81831037,"identity":"000caf17-194b-458a-bc60-20a0cac77684","added_by":"auto","created_at":"2025-05-02 13:42:05","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4727953,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.Fig.1bMitochondria48h.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6502413/v1/60e8eca87695ff46ddcf3229.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eMitochondrial transplantation improves bioenergetics and neurite outgrowth in disease-associated P301Ltau-expressing neuronal cells\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNeurodegenerative diseases are predominantly age-related and constitute a significant public health concern. These conditions are characterized by progressive degeneration and loss of neuronal cells in the central and peripheral nervous systems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Tauopathies belong to this group of neurodegenerative diseases. Notable examples include Alzheimer's disease (AD), fronto-temporal dementia (FTD), progressive supranuclear palsy, and amyotrophic lateral sclerosis. The tau protein, which is encoded by the MAPT gene (Microtubule Associated Protein Tau) on chromosome 17, plays a crucial role in the stabilization, assembly, and function of microtubules [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Moreover, this protein facilitates several essential functions in neuronal cells, including axonal transport, neurotransmission, and cell polarity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, in a pathological context, tau protein undergoes an abnormal hyperphosphorylation, resulting in intracellular tau accumulation and neurofibrillary tangle formation in the brain. The mechanisms underlying tau-induced neuronal dysfunction are not fully understood.\u003c/p\u003e \u003cp\u003eStudies have demonstrated that tau protein exhibits abnormal interactions with mitochondria, disrupting their cell functions and contributing to neuronal death and loss [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Mitochondria, also known as the \"powerhouses of the cell,\" are essential organelles contributing to vital cellular functions. They are not only required to ensure the production of the primary cellular energy through the generation of adenosine triphosphate (ATP) but they are also involved in a wide range of functions, such as calcium buffering and metabolite synthesis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Mitochondria are essential in neurons that require approximately 15% of the total body's energy to ensure processes such as neurotransmitter release, action potential, and synaptic plasticity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Given the pivotal role of mitochondria in these post-mitotic and highly differentiated cells, the impact of mitochondrial defects on brain functions is a salient consideration. Indeed, in tauopathies, the abnormal interaction of mitochondria with tau protein leads to impairment in mitochondrial morphology, like mitochondria swelling, and a decrease in their bioenergetic functions, such as impaired ATP production and an increase in reactive oxygen species (ROS) generation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Furthermore, mitochondrial dynamics, particularly the balance between fusion and fission processes, are altered, resulting in an impairment of the mitochondrial recycling system, also known as \"mitophagy, which in turn leads to increased cellular stress [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These mitochondrial impairments impact several cellular functions, including neuroplasticity with a deficit observed in the neurite outgrowth process. Indeed, the hyperphosphorylated tau protein cannot stabilize the microtubules correctly, which is necessary for the neurite\u0026rsquo;s elongation. Besides, it has been shown that the physiological tau protein can potentiate some pathways involved in neurite elongation initiation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It is widely recognized that these mitochondrial dysfunctions often precede the cognitive deficits observed in neurodegenerative diseases [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Neurons, as post-mitotic cells, cannot divide and replenish their supply in mitochondria [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Therefore, neuronal mitochondria represent an interesting target for therapeutic intervention. Conventional therapeutic approaches usually consist in improving mitochondrial bioenergetic functions and decreasing mitochondrial oxidative damage. A new approach consists of using exogenous healthy mitochondria as a treatment itself. This approach is called here mitochondrial transplantation.\u003c/p\u003e \u003cp\u003eThis innovative technique involves the isolation of mitochondria from healthy donor cells and their subsequent transfer into recipient cells that exhibit mitochondrial dysfunctions. This approach has been extensively documented in the context of cardiac diseases, where mitochondria also play a crucial role, as evidenced by the research conducted by the group of James McCully (Harvard Medical School, Boston, USA). They have demonstrated the efficacy of mitochondrial transplantation in enhancing cellular viability following ischemia-reperfusion injury in the heart [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. An increasing number of research groups are exploring the potential of mitochondrial transplantation to treat disorders in various organs, including the kidneys, liver, and brain [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Mitochondrial transplantation has been tested in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e settings for several brain disorders, including traumatic brain injury, cognitive deficit, neurodegenerative diseases, and brain cancer (reviewed in [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]). Overall, encouraging results are observed after mitochondrial transplantation, with notably an increase in mitochondrial functions, cellular viability, and an improvement of cognitive performance [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, no research has been conducted on the impact of mitochondrial transplantation in the context of tauopathies until today. To investigate this novel aspect, a cellular model (SH-SY5Y cells) overexpressing the P301L-tau mutation was used in the present study. This model has been previously characterized in our laboratory and has been shown to exhibit abnormal tau hyperphosphorylation, accompanied by bioenergetic deficits and mitochondrial impairments [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. It has been demonstrated that physiological transfers between glial cells and neurons occur in physiological conditions, leading to neuroprotective effects and improvement of cellular functions. Therefore, an astrocytic cell line named \u0026ldquo;A172\u0026rdquo; was used as a donor of healthy mitochondria for the transplantation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe hypothesized that mitochondrial transplantation could enhance cellular viability, neurite outgrowth, and bioenergetic functions in cells overexpressing disease-associated tau protein. In the first step, we investigated the effect of mitochondrial transplantation on a healthy cellular model to ascertain the optimal parameters. Namely, we assessed the impact of mitochondrial transplantation on cell viability, ATP levels, mitochondrial membrane potential, cellular oxygen consumption rate, and reactive oxygen species (ROS) levels. In the next step, we used the same approach and bioenergetic readouts with the \"P301L-tau\" cellular model of tauopathies. Finally, the study was completed with a neurite outgrowth investigation on the healthy and the P301L cells to assess the impact of mitochondria transplantation on neuronal morphology and differentiation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents/Chemicals\u003c/h2\u003e \u003cp\u003eAll the materials used for the study are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of materials and reagents used for the experiments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReagent/ressource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReference or source\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIdentifier or catalog number\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eChemical, enzymes, materials\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAccutase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInnovative Cell Technologies, Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAT-104\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATPlite 1step Luminescence Assay System\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRevvity Healthy Sciences Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimary antibody anti-\u0026szlig;3 tubulin chicken\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eab41489\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSecondary antibody anti-chicken coupled Alexa Fluor 488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eab150173\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlasticidin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvivogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnt-bl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBrdU kit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMerck\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQIA58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBSA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA9647\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB-27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGibco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17504001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell scraper\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSarstedt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e83.3951\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCellTracjer Blue CMAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC2110\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCulture dishes 10 cm\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSarstedt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e83.3902.300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCulture dises 20 cm\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSarstedt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e83.39.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDAPI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMerck\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10236276001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDHE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD11347\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD6429\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMSO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e276855\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEGTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermoscientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e409910250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFetal bovine serum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorning\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35-079-CV\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFilter 40 \u0026micro;m mesh size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePluriSelect\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43-57040-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFilter 10 \u0026micro;m mesh size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePluriSelect\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43-50010-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG418\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e108321-42-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlutamate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eG1626\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlutaMax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35050087\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBSS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGibco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14065049\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHorse serum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBioConcept\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2-05F00-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK-HEPES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResearch Organics Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6003H\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP5655\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMalate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMannitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM4125\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMgCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM4880\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMitoSOX Red\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM36008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMerck\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM2128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeurobasal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGibco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21103-049\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVWR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e392\u0026ndash;0440\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePFA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP6148\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePenicillin/Streptomycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBioconcept\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4-01F00‐5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRetinoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR2625\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSaccharose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEurobio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e018363\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubtilisin A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedChem Express\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHY-E70076\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSuccinate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS2378\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTMRM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFluka\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87919\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlack 96 well plates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreiner\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e655090\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWhite 96 well plates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreiner\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e165306\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClear 96 well plates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFalcon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e353072\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClear 12 well plate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFalcon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e353043\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCollagen I precoated slide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNeuvitro\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGC-18-1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eExperimental models\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA172 untransfected\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCRL-1620\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP301L-Tau-GFP transfected SH-SY5Y neuroblastoma cells (Human)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThe G\u0026ouml;tz laboratory\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHealthy SH-SY5Y neuroblastoma cells transfected with GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCRL-2266\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDevices and software\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEclipse Ti2 Nikon Microscope\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNikon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMultiplate reader Cytation 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAgilent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMultiplate reader Cytation 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAgilent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeurites Outgrowth Module\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAgilent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResipher Lucid Lab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLucid Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://lucidsci.com\u003c/span\u003e\u003cspan address=\"https://lucidsci.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTake3 Microvolume plate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAgilent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFiji (Fiji is just ImageJ) 2.16.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNIH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/software/fiji/\u003c/span\u003e\u003cspan address=\"https://imagej.net/software/fiji/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrism version 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGraphPad\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.graphpad.com/features\u003c/span\u003e\u003cspan address=\"https://www.graphpad.com/features\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuygens Deconvolution Software version 14.10.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eScientific Volume Imaging\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://svi.nl/Huygens-Software\u003c/span\u003e\u003cspan address=\"https://svi.nl/Huygens-Software\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImaris version 9.9.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOxford Instruments\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imaris.oxinst.com\u003c/span\u003e\u003cspan address=\"https://imaris.oxinst.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell lines\u003c/h3\u003e\n\u003cp\u003eThe human neuroblastoma cell line SH-SY5Y (ATCC, CRL-2266) was used as the recipient cells for the mitochondrial transplantation procedure. These cells have been documented in the scientific literature and are widely used as neuronal models in neuroscience research.\u003c/p\u003e \u003cp\u003eAs a cellular model for tauopathies, we used human neuroblastoma SH-SY5Y cells stably overexpressing the P301L mutation (\u0026ldquo;P301L cells\u0026rdquo;) on the MAPT gene of chromosome 17 and tagged with a green fluorescence protein (GFP). This model was developed and generously provided by Professor J\u0026uuml;rgen G\u0026ouml;tz's laboratory at the University of Queensland (Brisbane,Australia) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The control cells consist in SH-SY5Y cells expressing the GFP-vector only (\"vector cells\"). For both cell models (vector and P301L cells), 4.5 \u0026micro;g/ml of blasticidin was added to the culture medium to maintain a stable expression of the plasmids.\u003c/p\u003e \u003cp\u003eSince intercellular mitochondria transfer naturally occurs between neurons and astrocytes, resulting in enhanced neuronal functions and neuroprotection [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], a human astrocytic cell line A172 (ATCC, CRL-1620) was used as the mitochondria donor to provide healthy mitochondria for the bioenergetics experiments. In parallel, A172 cells stably expressing the red fluorescent protein (RFP) tagged to mitochondria were used, allowing the visualization of the transplanted astrocytic mitochondria in the neuronal cells for the microscopic experiments.\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eSH-SY5Y cells were cultured in a growing medium composed of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% penicillin and streptavidin, 1% glutamax, 10% fetal bovine serum (FBS), and 5% horse serum (HS). The selection antibiotic (blasticidin) was added during the medium change. The cells were cultivated in 10 cm\u003csup\u003e2\u003c/sup\u003e culture dishes containing 10 ml of growing medium.\u003c/p\u003e \u003cp\u003eA172 cells were cultured in DMEM supplemented with 1% penicillin/streptavidin, 1% glutamax, and 10% FBS. These cells were maintained in 10 cm\u003csup\u003e2\u003c/sup\u003e culture dishes and replated in a 20 cm\u003csup\u003e2\u003c/sup\u003e before the mitochondrial isolation.\u003c/p\u003e \u003cp\u003eThe cells were detached from the culture dishes using accutase and split twice weekly once they reached approximately 80% confluency. The cells were subsequently stored in an incubator maintained at 37\u0026deg;C and a CO\u003csub\u003e2\u003c/sub\u003e concentration of 5%.\u003c/p\u003e\n\u003ch3\u003eCellular differentiation\u003c/h3\u003e\n\u003cp\u003eTo be as close as possible to a neuronal phenotype, SH-SY5Y vector and P301L cells were differentiated into neuronal cells. Cells were plated in a 96-well plate format, and the growing medium was exchanged with 100 \u0026micro;L of differentiation medium 24 hours after the seeding of cells. The differentiation medium comprised Neurobasal medium, 1% penicillin and streptavidin, 2% B-27, and 10 \u0026micro;M retinoic acid. A washing step with phosphate-buffered saline (PBS) was performed before the incubation with the differentiation medium to ensure the complete removal of FBS and HS. The cells were maintained in the differentiation medium for three days. Subsequently, half of the volume of the differentiation medium was replaced to refresh the medium before the mitochondrial transplantation.\u003c/p\u003e\n\u003ch3\u003eMitochondrial isolation\u003c/h3\u003e\n\u003cp\u003eThe protocol for mitochondrial isolation has been previously established by Preble et al. in 2014 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. All procedures were conducted in a controlled, sterile environment. For each mitochondrial isolation, a full dish of 20 cm\u003csup\u003e2\u003c/sup\u003e of A172 cells was prepared. A homogenization buffer was prepared, composed of 300 mM saccharose, 10 mM K-HEPES, and 1 mM EGTA, which were diluted in ultrapure water. The pH was adjusted to 7.2 with a solution of 1 M NaOH. The solution was filtered under sterile conditions using a 0.2 \u0026micro;m filter and stored at 4\u0026deg;C until use.\u003c/p\u003e \u003cp\u003eMedium was removed from the dish containing A172 cells, followed by one wash with PBS. Then, 1 ml of homogenization buffer was added to the dish, and the cells were mechanically harvested using a cell scraper. The cells were centrifuged at 4\u0026deg;C for 5 minutes at 700 x g. Next, the cell pellet was suspended in 2 ml of homogenization buffer, and cells were lysed using a Potter grinder. A solution containing 4 mg/ml Subtilisin A was added to the tube and incubated on ice for 10 minutes. Filtration steps were then performed to remove the majority of cell debris. First, cell lysate was filtered using a 40 \u0026micro;m mesh size filter. Then, 500 \u0026micro;l of 20 mg/ml BSA (bovine serum albumin) was added to the lysate before a second filtration step with a new filter with a 40 \u0026micro;m mesh size, followed by a third filtration with a mesh size of 10 \u0026micro;m. Finally, the filtrated cell lysate was centrifugated at 4\u0026deg;C for 10 minutes at 10,000 x g. The final pellet was suspended in 200 \u0026micro;l of mitochondrial assay solution (MAS) and stored on ice until the transplantation step. Of note, the MAS is composed of 200 mM mannitol, 70 mM saccharose, 10 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2 mM K-HEPES, 1 mM K-EGTA, and 0.2% BSA in 200 ml of ultrapure water [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The MAS pH is adjusted to 7.2, and the solution is frozen at -20\u0026deg;C for extended periods.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of mitochondrial concentration\u003c/h2\u003e \u003cp\u003eThe concentration of isolated mitochondria was determined by conducting a protein assay with the Cytation 3 and the Take3 Microvolume plate. The software utilized for data analysis was Gen3.16. The protein absorption was measured at a wavelength of 280 nanometers (nm), and the MAS was employed for the blank measurement. This approach is characterized by its reliability, efficiency, and speed, thereby ensuring optimal conditions for preserving the integrity of isolated mitochondria before transplantation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMitochondrial transplantation\u003c/h3\u003e\n\u003cp\u003eIsolated mitochondria were kept on ice in the MAS for less than 1 hour. The acceptor cells (SH-SY5Y cells) were incubated with the isolated mitochondria (mitochondria-transplanted group), while the MAS alone was used as the control condition. A final volume of 10 \u0026micro;l of isolated mitochondria was added in the 100 \u0026micro;l of medium in 96-wells plate containing neuroblastoma cells. The final concentrations of isolated mitochondria that were tested are 5 \u0026micro;g/ml, 10 \u0026micro;g/ml, 25 \u0026micro;g/ml, and 50 \u0026micro;g/ml. The selection of these concentrations was guided by the findings of Shi et al., who conducted a study to ascertain the most efficacious concentrations for SH-SY5Y cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The cell plate was then incubated at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 or 48h.\u003c/p\u003e\n\u003ch3\u003eValidation of the entry of isolated mitochondria into recipient cells\u003c/h3\u003e\n\u003cp\u003eVector cells were plated in a 12-well plate on collagen I pre-coated coverslipes at a density of 5 x 10\u003csup\u003e4\u003c/sup\u003e cells/well. Cells were then differentiated by following the differentiation protocol previously described. To investigate whether isolated mitochondria can enter into cells, A172 cells expressing the RFP-tagged mitochondria (mitoRFP) were used as a mitochondrial source. After 24 and 48 hours of co-incubation with isolated mitochondria, Vector cells were incubated with 5 \u0026micro;M of CellTracker Blue dye for 30 minutes at 37\u0026deg;C to visualize the cells\u0026rsquo; area. Then, cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature (RT). Pictures were captured using an Eclipse Ti2 widefield microscope (Nikon). Images were subjected to a deconvolution step using the Huygens Deconvolution Software. Subsequent analysis was conducted using the Fiji and Imaris softwares.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCellular viability assay\u003c/h2\u003e \u003cp\u003eThe cellular viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. This colorimetric test reflects the metabolic activity of living cells and is interpreted as an indicator of cellular viability and toxicity. The experiment was conducted by seeding 1.5 x 10\u003csup\u003e4\u003c/sup\u003e SH-SY5Y cells per well in clear 96-well plates. The MTT was first diluted in the culture medium at 5 mg/ml. Then, the diluted MTT was added to the SH-SY5Y cells and incubated for 2 hours at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. The medium was carefully removed from the wells and substituted with 150 \u0026micro;L of dimethyl sulfoxide (DMSO). The plate was then placed on a shaker at 250 rpm for 15 minutes. The resulting absorption was measured at a wavelength of 550 nm using the Cytation 3 Cell Imaging Multi-mode Plate Reader (BioTek, Agilent, Switzerland). This test was conducted 24h and 48h after the mitochondrial transplantation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eATP levels\u003c/h2\u003e \u003cp\u003eThe test was conducted using the \"ATPlite 1step Luminescence Assay System\" kit. The assay is based on a bioluminescence reaction between an enzyme, luciferase, and a substrate, luciferin. This reaction requires the presence of ATP, with the emitted light being proportional to the level of ATP in each well. The cells were seeded in white 96-well plates (1.5 x 10\u003csup\u003e4\u003c/sup\u003e cells/well) to optimize the signal in the plate reader. An ATP standard (ranging from 0 to 20 \u0026micro;M) was prepared in duplicate to calculate the ATP concentration in our samples. To initiate the enzymatic reaction, 100 \u0026micro;l of ATP substrate was added to each well. Then, the plate was placed on the shaker at 200 rpm for 2 min, and the bioluminescence signal was read with the Cytation 3 instrument. The tests were conducted at 24h and 48h post-transplantation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Mitochondrial Membrane Potential\u003c/h2\u003e \u003cp\u003eThe mitochondrial membrane potential (MMP) was determined using the TMRM dye (tetramethylrhodamine methyl ester). The dye\u0026rsquo;s capacity to enter into the mitochondrial matrix is proportional to the MMP.\u003c/p\u003e \u003cp\u003eTo assess the membrane potential of mitochondria freshly isolated from A172 cells, and to ascertain their bioenergetic activity, two tubes containing 100 \u0026micro;g/ml of isolated mitochondria in MAS were prepared and incubated with 0.4 \u0026micro;M of TMRM for 30 minutes at 37\u0026deg;C. Subsequently, the samples were centrifuged at 10,000 x g for 10 minutes at room temperature. Then, the mitochondria pellet from the first tube was resuspended in the MAS, while the second pellet was resuspended in the MAS supplemented with 0.05 mM Glutamate, 8 mM Succinate, and 5 mM Malate which are substrates required for the electron transport chain activity. Finally, 50 \u0026micro;l of samples were added to black 96-well plates, and the emitted fluorescence (Excitation/Emission: 550/580 nm) was detected using the Cytation 3.\u003c/p\u003e \u003cp\u003eTo assess the effects of mitochondrial transplantation on the MMP of neuronal cells, SH-SY5Y cells were seeded in black 96-well plates at a density of 1.5 x 10\u003csup\u003e4\u003c/sup\u003e cells/well. The measurement of the MMP was conducted at 24h and 48h following the transplantation with isolated mitochondria. The TMRM dye was added to each well at a final concentration of 0.4 \u0026micro;M. The plate was then placed in the dark on a shaker at 100 rpm for of 30 minutes. Two washing steps were then performed with HBSS, and the fluorescence intensity (Excitation/Emission: 550/580 nm) was measured with the Cytation 3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of superoxide anion radical levels\u003c/h2\u003e \u003cp\u003eThe total and mitochondrial superoxide anions levels were measured using the DHE (dihydroethidium) and MitoSOX Red dyes, respectively. The cells were plated in black 96-well plates at a density of 1.5 x 10\u003csup\u003e4\u003c/sup\u003e cells/well. The superoxide anion levels were determined at 24h and 48h post-transplantation. The cells were incubated with 10 \u0026micro;M of DHE for 20 minutes in the dark at room temperature or 50 \u0026micro;M of MitoSOX red for 90 minutes at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were then washed twice with HBSS and the fluorescence intensity (Excitation/Emission: 531/595 nm), which is proportional to the total or mitochondrial superoxide anion levels, was detected with the Cytation 3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCellular respiration measurement\u003c/h2\u003e \u003cp\u003eThe Resipher device was utilized to assess cellular respiration over time. This device allows the measurement of oxygen consumption rate (OCR) directly in the cell incubator in real time (Lucid Scientific [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]). The Resipher device was affixed magnetically to a designated lid containing sensors placed on 96-well plates with 1.5 x 10\u003csup\u003e4\u003c/sup\u003e SH-SY5Y cells/well. The medium volume of 90 \u0026micro;l per well was selected based on the recommendations of Lucid lab to ensure optimal measurement conditions. The experimental protocol commenced 24h prior to transplantation and concluded 48h post-transplantation. The OCR values were extracted on the Lucid Scientific platform.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBrdU assay\u003c/h2\u003e \u003cp\u003eTo ascertain that the observed effect after the mitochondrial transplantation is not attributable to a proliferative effect, a BrdU (Bromodeoxyuridine) kit was used. In brief, the BrdU (1:2000) was incubated with undifferentiated SH-SY5Y cells overnight to allow one complete cycle of the cell division process. The following day, the sample underwent fixation and denaturation, followed by anti-BrdU immunostaining. The anti-BrdU secondary antibody is coupled with the horseradish peroxidase (HRP) enzyme, producing a colored product. The reaction is then halted using a solution provided in the kit. The absorbances were measured at 450 nm and 540 nm using the Cytation 3. The final values correspond to the difference between the absorbances at 450 nm and 540 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eNeurite outgrowth measurement\u003c/h2\u003e \u003cp\u003eThe impact of mitochondrial transplantation on neuronal plasticity was assessed using the Agilent BioTek Gen5 neurite outgrowth module. Cells were seeded in black 96-well plates and maintained in the differentiation medium for three days. The differentiation medium comprised Neurobasal, 1% penicillin, 1% streptavidin, 2% B27, and 10 \u0026micro;M retinoic acid. Thereafter, the differentiation medium was replaced with Neurobasal medium containing 1% penicillin and streptavidin, 2% B27, and different treatment conditions: MAS only (control), isolated mitochondria, or 50 ng/ml nerve growth factor (NGF) as a positive control.\u003c/p\u003e \u003cp\u003eAfter 48h of treatment, an immunostaining with anti-\u0026szlig;3 tubulin was performed. Cells were fixed with 4% PFA for 10 minutes at RT and permeabilized with 0.15% Triton in PBS for 15 minutes at room RT. Samples were incubated with 2% BSA in PBS to block unspecific sites for 1 hour. The primary antibody (anti-\u0026szlig;3 tubulin, chicken, 1:1000) was incubated overnight at 4\u0026deg;C. The secondary antibody (anti-chicken and preabsorbed with Alexa Fluor 488, 1:500) was subsequently incubated for 1 hour in the dark at RT. Finally, the cells were incubated with a nuclear staining solution (DAPI, 1:1000) for 5 minutes at RT. The samples were then preserved in PBS and stored at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eData from the neurite outgrowth experiments were obtained with the Cytation 5 Cell Imaging Multi-mode Plate Reader (BioTek, Agilent, Switzerland). Pictures were submitted to a preprocessing step with the predefined parameters in the software for both channel DAPI and GFP (rolling ball diameter: 138 \u0026micro;m). Then, a deconvolution step was applied to the preprocessed images (Point spread function: \u0026ldquo;Auto, based on objective\u0026rdquo;; iterations number: 5). The neurite outgrowth module was applied to the deconvolved images with experimental setting established for vector and P301L cells. The parameters collected for this study were: the average neurite length (the total skeleton length of all neurites divided by the soma count), the average neurite count per cell (number of neurites connected to the identified soma), average neurites branches per cell (count the number of ending branches connecting to neurites divided by the soma count) and the neurite thickness ( correspond to the total neurite area divided by the total neurite length, in \u0026micro;m).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll results were exported to Microsoft Excel 2019\u0026reg; and analyzed using GraphPad Prism 10\u0026reg; software. The results, depicted as boxes and whisker plots, indicate the median value, the mean, and the distribution of values with the minimal and maximal points (\"whiskers\"). The values are expressed as percentages of the control (MAS) condition. To ensure the reliability and reproducibility of the experimental results, each experiment was performed at least three times.\u003c/p\u003e \u003cp\u003eThe statistical analysis employed was One-way ANOVA with a post-hoc Dunnett's multiple comparison test versus the control condition or Two-ways ANOVA with a post-hoc Tuckey's multiple comparison test. Results were considered statistically significant if p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Establishment of the optimal mitochondrial transplantation parameters on the SH-SY5Y cellular model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAstrocytic mitochondria were freshly isolated from the A172 cells. Microscopic experiments were performed to assess the ability of isolated mitochondria to enter into recipient cells. As shown in the microscopy pictures and 3D representations in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, the isolated mitochondria are present in the recipient cells after 24h and 48h of incubation (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eTo ensure that the isolated mitochondria were still healthy and functional, we first assessed whether their mitochondrial membrane potential (MMP) was preserved and the electron transport chain (ETC) activity was still efficient. We observed that the TMRM dye was still capable of entering into isolated mitochondria and emitting a fluorescent signal, which reflected a polarized membrane potential (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). The addition of ETC substrates (glutamate, succinate, and malate) resulted in an augmentation of MMP in comparison without substrates. These results indicated that the structural and functional integrity of mitochondria was preserved after the isolation process.\u003c/p\u003e\n\u003cp\u003eNext, we determined the optimal concentration and incubation time for the transplantation of isolated mitochondria. Initial trials were conducted on healthy and undifferentiated SH-SY5Y cells. Based on the methodologies outlined by Shi et al. [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e], a range of concentrations were selected for subsequent experimentation: 5, 10, 25, and 50 \u0026micro;g/ml, and two time points: 24h and 48h.\u003c/p\u003e\n\u003cp\u003eFirst, we assessed whether the treatment with isolated mitochondria was toxic to cells. To do so, we performed an MTT viability assay. No significant difference was observed between the control (no mitochondria) and all the tested conditions (from 5 \u0026micro;g/ml to 50 \u0026micro;g/ml) at 24h after mitochondria transplantation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). Although mitochondrial transplantation did not increase cell viability, it was not toxic for the cells. In parallel, we checked the effect of the treatment on the metabolic activity of the cells using an ATP assay. The same experimental conditions were used previously for the MTT assay. A dose-response curve was observed after one day of transplantation, with a significant increase for the treated conditions compared to the control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). Especially, a\u0026thinsp;+\u0026thinsp;9% and +\u0026thinsp;19% increase in ATP levels were observed with the 25 and 50 \u0026micro;g/ml, respectively. These two treatment concentrations were selected to continue the screening phase. We confirmed the MTT and ATP results 48h after transplantation. As observed on the graphs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef), the same trend was observed after two days of treatment, with no difference between the control and treated conditions regarding the cellular viability (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee) but an increase in cellular metabolic activity (+\u0026thinsp;10% and +\u0026thinsp;12% increase for the 25 and 50 \u0026micro;g/ml conditions, respectively) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef).\u003c/p\u003e\n\u003cp\u003eSince it is known that ATP production is closely related to the MMP, this parameter was measured using the TMRM dye. In comparison with the control group, no difference was observed after 24h of treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg). A significant increase in MMP was detected after 2 days of incubation for both treated conditions compared to the control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eh). At this time point, MMP is statistically higher in the 50 \u0026micro;g/ml condition (+\u0026thinsp;96% increase versus the control) than in the 25 \u0026micro;g/ml condition (+\u0026thinsp;48% increase versus the control). A BrdU assay was performed to ensure that these beneficial effects were not due to an increase in cell proliferation after treatment. No variation in cell proliferation was observed at 24h post-transplantation for both tested concentrations (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ei). In addition, a decrease in this parameter (\u0026minus;\u0026thinsp;9%) was observed after treatment with 25 \u0026micro;g/ml of isolated mitochondria for 48h (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ej). Therefore, all the beneficial effects observed in the previous data were due to mitochondrial transplantation and not to cell proliferation.\u003c/p\u003e\n\u003cp\u003eBased on all these results, the 25 \u0026micro;g/ml mitochondria concentration was selected for subsequent analysis. Indeed, this concentration showed a beneficial effect on cell functions with no obvious difference with the 50 \u0026micro;g/ml treatment condition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Isolated mitochondria improve bioenergetic functions and reduce the oxidative stress of the vector SH-SY5Y cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo get closer to a neuronal model, the second part of the experiments was performed on differentiated and healthy SH-SY5Y cells (\u0026quot;vector\u0026quot; cells). Based on the previous results from the screening phase, the optimal treatment concentration was 25 \u0026micro;g/ml. The same bioenergetic assays were performed on this cell model, with, in addition, the assessment of superoxide anion radical levels and cellular respiration at 24h and 48h post-transplantation.\u003c/p\u003e\n\u003cp\u003eThe potential toxicity of the treatment was assessed at 24h and 48h post-transplantation. We observed an increase in viability compared to the corresponding control condition of +\u0026thinsp;28% and +\u0026thinsp;34%, respectively. However, no difference was observed between the treated condition at 24h and 48h (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). An increase in cellular metabolism was also observed, with +\u0026thinsp;25% and +\u0026thinsp;45% increase in ATP production after treatment at 24h and 48h, respectively. For this parameter, the production of ATP is significantly higher at 48h post-transplantation compared to the treated group at 24h (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). In parallel, MMP was increased of +\u0026thinsp;24% and +\u0026thinsp;31% after one and two days of treatment, respectively, showing a correlation with ATP levels. However, there was no difference between the treated condition at 24h and 48h after transplantation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003eWe next assessed whether the treatment\u0026apos;s effects were related to an increase in cellular respiration by measuring the oxygen consumption rate (OCR) in the recipient cells. Compared to the corresponding control condition, an increase in OCR was observed at 24h (+\u0026thinsp;42%) and 48h (+\u0026thinsp;52%) after transplantation. This increase appeared stable over time, as there was no significant difference between the treated conditions at both time points (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003eThe main risk in improving the bioenergetic functions of cells is the parallel increase in the production of reactive oxygen species (ROS). Therefore, cellular and mitochondrial superoxide anion radical levels were measured using fluorescent dyes. Surprisingly, mitochondrial superoxide anion levels were decreased at 24h (-14%) and 48h (-16%) after treatment. No difference was observed between the two time-points (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee). Therefore, this effect is maintained over time. In addition, total superoxide anion radical levels were decreased at 48h (-12%) after transplantation with isolated mitochondria (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e\n\u003cp\u003eThus, mitochondrial transplantation improves the cellular viability and bioenergetic functions of differentiated vector cells. In addition, a decrease in oxidative stress was observed after treatment. Most of the results appeared at 24h and 48h post-transplantation, which means that the transplantation might have a positive effect in the long term.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Mitochondrial transplantation improves the bioenergetics functions in P301L SH-SY5Y cells and reduces the mitochondrial superoxide anions levels.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince the results of the tests with the control (vector) cells were maintained at 24h and 48h, the experiments with the P301L SH-SY5Y cells were performed at the same time points. The cellular deficits of the P301L model compared to the control cells have been previously demonstrated in our laboratory [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eCell viability was improved at 48h post-transplantation with an increase of +\u0026thinsp;22% compared to the untreated condition. No significant differences were observed at 24h (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). In parallel, an increase in ATP production was observed at 24h (+\u0026thinsp;17%) and 48h (+\u0026thinsp;31%) compared to the control condition. This time, the effect of the treatment was higher at 48h than 24h (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). The MMP was significantly increased in P301L 24h post-transplantation, however this effect was lost after 48 hrs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). Cellular respiration was also examined in P301L cells. An increased OCR was observed after treatment at 24h (+\u0026thinsp;18%) and at 48h (+\u0026thinsp;36%) compared to the corresponding control condition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed). As for the ATP production, the respiratory capacity of the cells seemed to correlate with the incubation time of the isolated mitochondria with the P301L cells.\u003c/p\u003e\n\u003cp\u003eA previous study of our group has shown that ROS level is higher in the P301L cells than in the control cells [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. Thus, the impact of mitochondrial transplantation on oxidative stress is an essential question in this case. Therefore, total and mitochondrial superoxide anion radical levels were assessed in P301L cells after mitochondria transplantation. We observed a significant decrease in mitochondrial ROS levels at 24h (-13%) and at 48h (-9%) compared to the corresponding control condition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee). This effect appeared to be stable over time, with no statistical difference between the treated condition at either time point. However, no decrease in total ROS levels was observed after treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e\n\u003cp\u003eOverall, mitochondrial transplantation improved the cellular viability and bioenergetic functions of P301L cells after 24h and 48h of incubation. In addition, mitochondrial oxidative stress is reduced after treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Mitochondrial transplantation increases the neurite outgrowth in the vector and P301L cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe showed that mitochondrial transplantation improved the bioenergetic characteristics of the vector and P301L after 48h of incubation. Because mitochondrial bioenergetics is essential in building neuronal networks by providing the energy necessary for neurite formation, a morphological analysis of neurite outgrowth was performed on the vector and P301L cells after two days of treatment with mitochondria. The characterization of the neurite outgrowth parameters of the P301L cells in comparison with the vector is shown in the supplementary Fig.\u0026nbsp;2. In these experiments, nerve growth factor (NGF) was used as a positive control, and all the neurite outgrowth parameters were measured after 48h of treatment.\u003c/p\u003e\n\u003cp\u003eTo measure several parameters characterizing neurite outgrowth, we used the Cytation 5 (Agilent) and the \u0026quot;Neurite Outgrowth Module\u0026quot;, which allowed us to generate masks highlighting the soma and neurites of the neuronal cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef). Neurite outgrowth parameters were automatically calculated by the Gen5 imaging software.\u003c/p\u003e\n\u003cp\u003eThe vector and P01L cells treated with the positive control NGF presented an increase in the average neurite count (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eg), the average neurite branches (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eh), the average neurite length (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ei) and a decrease in neurite thickness (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ej), indicating an enhanced neuronal differentiation and plasticity.\u003c/p\u003e\n\u003cp\u003eIn the mitochondria-treated cell, we observed an increase in the average number of neurites and branches per cell in the vector (+\u0026thinsp;30% and +\u0026thinsp;19%, respectively) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec) and P301L cells (+\u0026thinsp;51% and +\u0026thinsp;50%, respectively) compared to the control condition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eg, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eh). In addition, neurite length was also increased after treatment for P301L cells (+\u0026thinsp;42%) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ei). Of note, the average neurite length observed after mitochondrial transplantation in P301L cells was as high as with the NGF treatment. In parallel, a decrease in neurite thickness was observed for both cell lines (-9% and \u0026minus;\u0026thinsp;13% for vector and P301L cells, respectively) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ej).\u003c/p\u003e\n\u003cp\u003eIn conclusion, mitochondrial transplantation positively impacted the neuronal differentiation process, especially in P301L, which presented an increase in every neurite outgrowth parameter.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we hypothesized that mitochondrial transplantation could improve the cellular viability, bioenergetic functions, and neurite outgrowth of healthy and tau mutant P301L cells.\u003c/p\u003e \u003cp\u003eIn the first step, we optimized the mitochondria isolation protocol and determined the optimal treatment concentration. Our data indicate that the protocol used for the mitochondrial isolation preserved mitochondrial function and integrity because the mitochondria were still bioenergetically active after the process. Moreover, we showed that the isolated mitochondria can enter the recipient cells. In line with that, a previous study from Shi et al., have shown that isolated mitochondria can enter the recipient cells after only 30 min of incubation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In our study, we observed that isolated mitochondria were present in the recipient cells after 24h and 48h of incubation.\u003c/p\u003e \u003cp\u003eIn the next step, we showed that the mitochondrial transplantation was not toxic for the neuronal cells, and that it had positive impacts on the respiratory chain activity by increasing the ATP and MMP levels. The BrdU assay revealed that the beneficial effects observed were not due to a proliferative effect of the treatment. Moreover, the decrease in cell proliferation observed after 48h of treatment in undifferentiated SH-SY5Y cells might be due to modulatory effects of the mitochondrial transplantation on neuronal differentiation. Indeed, the promoting effect of mitochondrial transplantation on neuronal differentiation was previously shown on schizophrenia-derived induced pluripotent stem cells (iPSCs) differentiated into glutamatergic neurons [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These data are in line with the results obtained in our neurite outgrowth experiments. After this screening phase, we selected the 25 \u0026micro;g/ml mitochondria concentration for further experiments on differentiated SH-SY5Y cells.\u003c/p\u003e \u003cp\u003eBecause undifferentiated SH-SY5Y cells are not representative of mature neurons, the cells were differentiated to get as close as possible to neuronal cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Mitochondria transplantation was performed after differentiation of the vector and the P301L SH-SY5Y cells. The cellular viability, ATP production, MMP levels, and cellular respiration were improved at 24h and 48h after the treatment for the vector cells. The effects of mitochondrial transplantation on the bioenergetic functions were more potent in the differentiated cells than the undifferentiated ones. One explanation could be that the bioenergetic metabolism of undifferentiated and differentiated cells is not the same. Indeed, the undifferentiated cells have a more glycolytic metabolism, while the differentiated cells primarily use the OXPHOS process [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. So, it might be possible that mitochondrial transplantation boosts the respiratory activity of the differentiated cells, with beneficial effects on the OCR, ATP production and MMP. In addition, a decrease in the total and mitochondrial superoxide anions levels was observed after the mitochondrial transplantation, in line with the increased cellular viability. These positive effects of the transplantation are reproducible between the 24h and the 48h timepoint, suggesting that treatment might be effective on the long-term. Longer treatment durations will have to be tested in future experiments to determine how long the effects of the transplantation are lasting.\u003c/p\u003e \u003cp\u003eThe second main aim of this study was to determine whether mitochondria transplantation could improve bioenergetic parameters in a cellular model of tauopathies overexpressing the P301L-tau mutation in the MAPT gene [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This model is mainly associated with frontotemporal dementia but is also widely used as a general model of tauopathies. In particular, the P301L-tau mutation is associated with abnormal hyperphosphorylation of the tau protein with neuronal, synaptic, and mitochondrial dysfunctions. The P301L-tau overexpressing SH-SY5Y cells have been previously characterized in our laboratory, and their bioenergetic deficits in comparison with the vector cells have been demonstrated [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Based on these previous findings, this study aimed to determine whether mitochondrial transplantation could improve bioenergetic functions in the P301L model. We observed increased cellular viability, ATP levels, MMP, and OCR after treatment with mitochondria. In addition, the positive effects were maintained for at least 48h.\u003c/p\u003e \u003cp\u003eRegarding the MMP, we observed that the mitochondria transplantation effect was lost in the P301L cells after 48h, as indicated by a decrease in the fluorescent signal compared to the 24h-treated group. Our hypothesis to explain this result is that a negative feedback loop might be activated in response to the high increase in ATP production after treatment. Indeed, the individual activity of each mitochondrion (the endogenous and the transplanted one) might possibly be lower with a MMP similar to the control condition. However, the global bioenergetic activity is higher than the control condition because mitochondria were added to the system, resulting in higher mitochondrial mass. This regulation can also play an \u0026ldquo;antioxidant role\u0026rdquo; in limiting the ROS production and oxidative stress in cells. On top of that, the transplanted mitochondria may have an antioxidant effect by decreasing the mitochondrial superoxide anion levels. It is known that the P301L cells have a higher level of ROS than the vector cells. Therefore, the healthy transplanted mitochondria may bring their own antioxidant system, including the manganese superoxide dismutase (Mn-SOD), which is highly present in the mitochondrial matrix [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. It has also been shown that the glutathione reductase levels were increased after the mitochondrial transplantation, suggesting that this enzyme might enhance the antioxidant system in the P301L cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMost of the bioenergetic properties of vector and P301L cells have been positively affected by mitochondrial transplantation. Therefore, it was interesting to focus also on morphological characteristics of the treated cells, such as the neurite outgrowth. It has been demonstrated in a hybrid model of mouse neuroblastoma and rat glioma NG108-15 overexpressing the cDNA of human tau441, that tau cells have a higher number of neurites, and these neurites were thicker than in the wild-type cells [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In line with that, our P301L-tau overexpressing cells present shorter neurites but more neurites per cell and more neurite branches than the vector cells. Indeed, the hyperphosphorylated tau protein is known to destabilize the microtubules, which are involved in the elongation of neurites. The higher number of neurites and branches in the P301L could be a way to compensate for the deficit in neurite length. It has been shown in a previous study that mitochondrial transplantation can enhance the neurite growth \u003cem\u003ein vitro\u003c/em\u003e on 6-OHDA-induced PC12 cells as a model of Parkinson's disease [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Our results are in line with these findings, as we observe an increase in the average number of neurites and branches per cell for the vector and P301L cells. The neurites length was also enhanced in the P301L cells, which suggests that the mitochondrial transplantation improves the neurites elongation capacity of these cells.\u003c/p\u003e \u003cp\u003eTherefore, mitochondrial transplantation improved bioenergetics functions and neuroplasticity process in the context of tauopathies, making this therapeutical approach relevant in tau-related neurodegenerative disorders.\u003c/p\u003e \u003cp\u003eBased on the pivotal role of mitochondria in neurons, this approach is now increasingly used in brain disorders studies, such as ischemia, Parkinson\u0026rsquo;s disease, and traumatic brain injury [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Several studies have shown improvement in bioenergetics functions, neuroprotective effects, neurogenesis, and anti-inflammatory response after mitochondrial transplantation in different \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Indeed, it has been demonstrated that mitochondrial transplantation improves the general mitochondria activity in cells with an increase in ATP production and a decrease in neuronal loss, a decreased inflammation in the hippocampus, and a reduction in ROS levels in a mouse model of AD induced by the injection of \u0026szlig;-amyloid in the intracerebroventricular site or the hippocampus [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In addition, in the same AD animal model, an increase in memory capacities and restoration of cognitive deficits have been shown after transplantation. In line, mitochondrial transplantation induced a decrease in the \u0026ldquo;depressive-like\u0026rdquo; behavior on a lipopolysaccharide-induced model of depression [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Improvement in the locomotor activity has also been observed after mitochondrial transplantation in a Parkinson\u0026rsquo;s disease mice model [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These effects were explained by the capacity of the transplanted mitochondria to module the gene expression in the recipient cells. Indeed, a few hours after the transplantation, genes related to mitochondrial function and neuronal repairment were upregulated, while the genes involved in the oxidative stress and apoptosis were downregulated [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The sirtuin 1 (SIRT1) pathway appeared to be recruited to ensure the positive effects observed by promoting BDNF production and autophagy in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, investigations on the underlying mechanisms and specific pathways recruited for the entry and the fate of mitochondria in the recipient cells are still missing [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMitochondrial transplantation has been more studied in the context of cardiac disease, where the first clinical study on pediatric patients has been realized [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These patients presented cardiac ischemia following a cardiac surgery event. Autologous mitochondria were isolated from the patient's accessible skeletal muscle, such as the rectus abdominis and the pectoralis, and were delivered by direct injection in the ischemic cardiac areas [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The authors did not observe adverse side effects in the short-term (including arrhythmia or scarring complications); four patients out of five had improved their ventricular function and no longer needed extracorporeal membrane oxygenation (ECMO) support. Therefore, this first clinical study has shown that this therapeutic strategy is promising for several diseases. The first clinical trial in the context of brain ischemia is currently underway (ClinicalTrials.gov: NCT04998357). As for the clinical trial in cardiac disease, this project aims to test the potential of autologous mitochondrial transplantation. The objective is to investigate whether the treatment could reduce the infarct volume. Mitochondria will be isolated from the muscle tissue close to the surgical access site. The treatment should be delivered in the brain artery via a micro-catheter during reperfusion. The presence or absence of side effects will be verified following the clinical trial scheduled for late 2026.\u003c/p\u003e \u003cp\u003eIn these two clinical trials, the source of mitochondria was the muscle of the patient itself. In our study, the A172 cells were chosen as mitochondrial sources because it has been shown \u003cem\u003ein vitro\u003c/em\u003e that astrocytes can naturally transfer their mitochondria to neurons under stress conditions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, the most relevant sources of mitochondria would be the same tissue as the targeted organ [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, the source of mitochondria to ensure the optimal effects of the transplantation is a relevant question, considering that their activity depends on their tissue origin [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Moreover, the delivery way of mitochondria is also a crucial part of the treatment. Indeed, a simple co-incubation, as realized in our study, is not feasible in a clinical context. Although, the intracerebral injection looked efficient in some studies on animal models, it might be too invasive to be applicable on human. Other studies used the intravenous injection, and they found the isolated mitochondria in different organs, including the brain, suggesting that isolated mitochondria can cross the blood-brain barrier [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The method of mitochondria delivery should be more specific to target the brain specifically. The nasal route represents an interesting option because it is non-invasive and does not require a high amount of mitochondria[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo our knowledge, no previous study has focused on the effects of mitochondrial transplantation in tauopathies. Our results have shown that mitochondrial transplantation improves bioenergetic functions and neurite outgrowth in a neuronal model of tauopathies. Therefore, further studies on the impact of mitochondrial transplantation on the other pathological features of tauopathies are needed. Deciphering the impact of mitochondrial transplantation on tau hyperphosphorylation, abnormal tau accumulation in neuronal cells, as well as on mitophagy and microtubule stabilization processes, might provide new insights supporting its potential as a therapeutic strategy for tauopathies. Further studies should be performed to verify our results on more advanced cellular models, like human iPSCs -derived neurons, and in animal models of tauopathy, considering all the preceding questions for clinical application.\u003c/p\u003e \u003cp\u003eTo conclude, mitochondrial transplantation represents a promising therapeutic approach to boost neuronal bioenergetics and alleviate abnormal tau-related neuronal impairments. In this approach, mitochondria are not a target for therapeutic intervention but represent the therapy themselves.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eATP: adenosine triphosphate\u003c/p\u003e\n\u003cp\u003eBrdU: bromodeoxyuridine\u003c/p\u003e\n\u003cp\u003eBSA: bovine serum albumin\u003c/p\u003e\n\u003cp\u003eDHE: dihydroethidium\u003c/p\u003e\n\u003cp\u003eDMEM: Dulbecco\u0026rsquo;s modified Eagle medium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDMSO: dimethyl sulfoxide\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFBS: fetal bovine serum\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGFP: green fluorescent protein\u003c/p\u003e\n\u003cp\u003eHRP: horseradish peroxidase\u003c/p\u003e\n\u003cp\u003eHS: horse serum\u003c/p\u003e\n\u003cp\u003eiPSCs: induced pluripotent stem cells\u003c/p\u003e\n\u003cp\u003eMAPT: microtubule associated protein Tau\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMAS: mitochondrial assay solution\u003c/p\u003e\n\u003cp\u003eMMP: mitochondrial membrane potential\u003c/p\u003e\n\u003cp\u003eMT: mitochondrial transplantation\u003c/p\u003e\n\u003cp\u003eMTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide\u003c/p\u003e\n\u003cp\u003eNGF: nerve growth factor\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOCR: oxygen consumption rate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePBS: phosphate buffered saline\u003c/p\u003e\n\u003cp\u003ePFA: paraformaldehyde\u003c/p\u003e\n\u003cp\u003eRFP: red fluorescent protein\u003c/p\u003e\n\u003cp\u003eROS: reactive oxygen species\u003c/p\u003e\n\u003cp\u003eRPM: revolutions per minute\u003c/p\u003e\n\u003cp\u003eRT: room temperature\u003c/p\u003e\n\u003cp\u003eTMRM: tetramethylrhodamine methyl ester\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the OPO Foundation, the Novartis Forschungsstiftung FreeNovation and the Dementia Research, Synapsis Foundation Switzerland (2022-PI05) and the B\u0026uuml;rgenstock Foundation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe are grateful to Olivier Buetzberger from the Agilent company for his help and availability during the installation and training of the neurite outgrowth module. We thank Fides Meier for the technical support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the OPO Foundation, the Novartis Forschungsstiftung FreeNovation and the Dementia Research, Synapsis Foundation Switzerland (2022-PI05) and the B\u0026uuml;rgenstock Foundation.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no competing interests. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: A.G.; Methodology: AB, AR; Data collection: A.B., A.G., A.R.; Formal analysis: A.B., A.G., A.R.; writing-original draft: A.B., A.G.; Funding acquisition: A.G.; Resources: A.G., A.E., Supervision: A.G., A.E., A.P. All authors have read and agreed to the published version of the manuscript. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe corresponding data presented in this study are available on Open Science Framework (https://osf.io/2pju6/?view_only=6f3983caf10546e881dbd2fa6cb70a98).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgnello L, Ciaccio M (2022) Neurodegenerative Diseases: From Molecular Basis to Therapy. Int J Mol Sci 23\u003c/li\u003e\n\u003cli\u003eAlexander JF, Mahalingam R, Seua AV et al. (2022) Targeting the Meningeal Compartment to Resolve Chemobrain and Neuropathy via Nasal Delivery of Functionalized Mitochondria. Advanced Healthcare Materials 11\u003c/li\u003e\n\u003cli\u003eChang J-C, Chao Y-C, Chang H-S et al. 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Translational Research 235:102-114\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Tauopathies, P301Ltau mutation, Mitochondria, Transplantation, Bioenergetic, Neurites","lastPublishedDoi":"10.21203/rs.3.rs-6502413/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6502413/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTauopathies are neurodegenerative diseases characterized by the abnormal accumulation of tau protein in neurons, leading to cognitive impairment. A common feature of these disorders is mitochondrial dysfunction, which results in bioenergetic deficits and contributes to neuronal death. As neurons have high energy demands, impaired mitochondrial function directly affects their viability and function. Thus, mitochondria represent an attractive target for neuroprotective strategies in tauopathies. Mitochondrial transplantation (MT) is an emerging therapeutic approach to restoring cellular bioenergetics. Although MT has shown promise in various models of brain diseases, its efficacy has not been evaluated in the context of tau-induced mitochondrial dysfunction. This study investigates the therapeutic potential of MT in a cellular model of tauopathy. Mitochondria were freshly isolated from astrocytic cells and transplanted into healthy SH-SY5Y neuroblastoma cells and SH-SY5Y cells overexpressing the P301L tau mutation. Bioenergetic and neuroplastic parameters were assessed 24 and 48h post-transplantation. Our results demonstrate that MT enhances cell viability, ATP production, mitochondrial membrane potential, and respiration in both healthy and tau-mutant neuronal cells. In addition, MT reduced mitochondrial superoxide anion levels and promoted neurite outgrowth in both cell lines. These findings suggest that MT is a promising therapeutic strategy for tauopathies. Importantly, this approach positions mitochondria not as a target but as the therapeutic agent itself. Further studies are warranted to advance mitochondrial transplantation toward clinical applications in tau-related neurodegenerative disorders.\u003c/p\u003e","manuscriptTitle":"Mitochondrial transplantation improves bioenergetics and neurite outgrowth in disease-associated P301Ltau-expressing neuronal cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-02 13:10:00","doi":"10.21203/rs.3.rs-6502413/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-29T16:07:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-28T09:45:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46425080200108380429876723813582900310","date":"2025-05-23T13:31:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-19T15:08:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88242688312004010747500983873304937420","date":"2025-05-19T01:43:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157406499712017732775650291427854636758","date":"2025-05-19T01:20:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-18T03:16:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-09T07:53:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-09T07:46:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2025-04-22T08:58:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a08ea5f9-c8b6-4def-aab0-b4e46ee083fd","owner":[],"postedDate":"May 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T16:08:28+00:00","versionOfRecord":{"articleIdentity":"rs-6502413","link":"https://doi.org/10.1007/s12035-025-05604-y","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2025-12-10 15:58:49","publishedOnDateReadable":"December 10th, 2025"},"versionCreatedAt":"2025-05-02 13:10:00","video":"","vorDoi":"10.1007/s12035-025-05604-y","vorDoiUrl":"https://doi.org/10.1007/s12035-025-05604-y","workflowStages":[]},"version":"v1","identity":"rs-6502413","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6502413","identity":"rs-6502413","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}